1. Hamilton County Coroner’s Laboratory DNA Standard Operating
Procedure
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
1.1 Goal: It is the goal of the laboratory’s program to:
1.1.1 Provide the users of the laboratory services access to DNA typing
of selected biological materials associated with official criminal
investigations using Polymerase Chain Reaction (PCR) analysis
methods.
Accept samples for DNA typing in accordance with case acceptance
criteria of the Hamilton County Coroner’s Laboratory. The criteria for
accepting DNA typing cases can be found in section 6.3.1 of this
manual.
1.1.2 Ensure the quality, integrity and accuracy of the DNA typing
data and its presentation through the implementation of a detailed
Quality Assurance (QA) program.
1.2 Objectives: It is the objective of the laboratory’s QA program to:
1.2.1 Monitor the analytical testing procedures and reporting of DNA
typing by means of Quality Control (QC) standards, proficiency
tests and audits.
1.2.2 Ensure that the entire DNA typing procedure is operating within
the established performance criteria and that the quality and validity
of the analytical data are maintained.
1.2.3 Ensure that problems are noted and corrective action taken and
documented.
1.3 Authority and Accountability
1.3.1 The Laboratory Director or Coroner’s Office Administrator shall
approve this policy and additions or deletions must meet their
approval before acceptance.
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1.3.2 The DNA Section Supervisor will be responsible for monitoring
the Quality Control/Quality Assurance Program.
1.3.3 DNA Analysts will be responsible for recording and maintaining
the QC records.
1.3.4 The QA guidelines prepared by the Technical Working Group on
DNA Analysis Methods (TWGDAM) provide a model for the Hamilton
County Coroner’s Laboratory DNA QA Program. The TWGDAM
guidelines do not, in their entirety, represent the laboratory’s QA
program. They have served as a starting point for the construction of
the QA program. Any supplements and revisions of the TWGDAM
guidelines and those of the DNA Advisory Board (DAB) will be
reviewed for possible incorporation into the QA program.
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2. Personnel
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
2.1 Job Descriptions
Job descriptions for all personnel including responsibilities and duties are
kept by the Laboratory Director and the Administrator.
2.2 Qualifications
The education, training, experience and qualifying criteria of technical
personnel within the DNA section is established. Members of the section
must demonstrate the ability to critically evaluate and interpret the
evidence, results and data. the minimum requirements for those individuals
are specified below.
2.2.1 Qualifying Procedure
It is highly desirable that these persons undergo a formal qualifying
procedure which reviews and documents that prerequisite criteria have
been satisfied prior to the assumption of duties. These criteria should
include:
2.2.1.1 Knowledge of the scientific principles, techniques and
literature of DNA typing.
2.2.1.2 Practical laboratory skills in the performance of DNA
analysis as demonstrated by observation and successful
analytical results.
2.2.1.3 Competency of individuals engaged in DNA analysis as
demonstrated by the successful completion of proficiency testing.
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2.2.2 Maintaining Qualification
The proficiency testing, performance and continuing education of
personnel is periodically reviewed as part of the laboratory’s overall
plan for quality assurance.
2.2.3 Technical Leader
2.2.3.1 Education
The analyst designated as technical leader must possess a
Master’s degree in a biological, chemical, or forensic science.
With undergraduate or graduate coursework in genetics,
chemistry, statistics, biochemistry, and molecular biology
(molecular genetics or recombinant DNA technology).
2.2.3.2 Training - Must have, at a minimum:
A. Training in the fundamentals of forensic biology.
B. Documented training in DNA analysis with individuals,
agencies, or other laboratories, in a program that includes
the methods, procedures, equipment and materials used in
forensic DNA analysis and their applications and limitations.
2.2.3.3 Experience
Minimum of five years of experience as a forensic science
analyst/examiner and a minimum of three years DNA
laboratory experience.
2.2.3.4 Continuing Education
Must stay abreast of developments within the field of DNA
typing by reading current scientific literature. Attendance at
seminars, courses or professional meetings is highly desirable.
Laboratory management provides the opportunity to comply
with the above requirements through the normal budgeting
process.
2.2.4 Examiner/Analyst
2.2.4.1 Education
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A laboratory analyst must possess a BS/BA degree in a
biological, chemical, or forensic science. Graduate or
undergraduate coursework in genetics, biochemistry, statistics,
and molecular biology (molecular genetics or recombinant DNA
technology) is recommended. An advanced degree is preferred.
2.2.4.3 Training - Must have, at a minimum:
A. Training in the fundamentals of forensic biology.
B. DNA analysis training with individuals, agencies, or
other laboratories having an established training program
and considerable experience in molecular biology and
forensic DNA casework.
2.2.4.3 Experience - Must at a minimum include:
A. One year forensic biology experience.
B. Prior to any casework examination or reporting, the
analyst must have a minimum of six months forensic
DNA laboratory experience.
2.2.4.4 Continuing Education
Must stay abreast of developments within the field of DNA
typing by reading current scientific literature, attending
seminars, courses or professional meetings. The distribution of
newly acquired knowledge to other analysts is recommended.
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3. Documentation
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
The Serology section of the Hamilton County Coroner’s Laboratory must
maintain documentation of all significant aspects of the DNA analysis
procedure, including any laboratory records that are pertinent to the analysis
or the interpretation of results. Documentation exists for the following topic
areas:
3.1 Analytical Methods and Procedures for DNA Typing
The information contained in this manual describes the protocol
currently used for the analytical testing of DNA.
A historical manual of all past analytical testing materials, procedures
and guidelines and all revisions thereof, including dates of such
revisions will be maintained.
3.2 Population Data Base
The Hamilton County Coroner’s Laboratory is not compiling a
population data base for the STR testing procedure. The FBI
population database included with the Popstats program is used to
calculate statistics for the casework in the section.
3.3 Quality Control of Reagents
Documentation of critical reagents is contained in the Quality Control
Manual. Critical Reagents for DNA are the Profiler Plus and Cofiler
kits. Documentation of their performance is located in the Critical
Reagent log and in the green file cabinet in the Serology section.
3.4 Case File/Notes
DNA analysis forms will be kept in every case file. These forms will
include the dates that the testing was performed, lot numbers of
reagents, membranes, and solutions. Electropherograms for the
samples in a case and the associated population statistics reports will
be kept in the case file. All notes and analysis forms within a case file
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will be kept in a logical order (i.e. extraction, quantitation,
electropherograms etc.) and will have each page numbered.
3.5 Data Analysis and Reporting
Specific requirements regarding data analysis and report writing are
included in Section 8 of this manual.
3.6 Evidence Handling Protocols
3.6.1 Evidence and samples from evidence are collected, received,
handled, sampled and stored so as to preserve the identity, integrity,
condition and security of the item.
3.6.2 Blood standards or buccal swab samples are required from the
victim, suspect, and from anyone else who may have contributed blood,
semen, saliva, or any other body secretion to the stain in question.
Blood standards should be collected in purple top (EDTA preservative)
tubes. Buccal swab samples should be collected, allowed to air dry,
and then submitted in properly marked paper envelopes.
3.7 Equipment Calibration and Maintenance Logs
Specific requirements are specified in Section 5 of this manual and also
in the Calibration and Maintenance Manual for the Serology section.
3.8 Proficiency Testing
Open and blind proficiency testing is recommended. Specific
requirements regarding proficiency testing at the Hamilton County
Coroner’s Office are specified in Section 9 of this manual and also in
the Laboratory’s Quality Manual. Blind proficiency tests are
coordinated by the Laboratory Director.
3.9 Personnel Training and Qualification Records
Training obtained by each analyst is kept in a log located in the
Serology section’s Training Manual.
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3.10 Method Validation Records
3.10.1 Copies of publications related to the procedure will be
circulated through the section and then held on file.
3.10.2 A new method must be tested using known samples and/or
proficiency samples. If significant modification has been made to the
analytical procedure, the modified procedure will be compared to the
original using identical samples.
3.10.3 Notebooks of all validation and research will be kept. These
will include all the procedures performed with the results of each
experiment.
3.10.4 Any changes in the protocol must be scientifically validated,
documented, and approved by the DNA typing personnel in the
Hamilton County Coroner’s Office before the changes are implemented
and a reviewed version of the DNA Profiling Protocol is generated.
3.11 Quality Assurance and Audit Records
Documentation supporting the QA program is included in the Quality
Control Manual and audits conducted in this lab are located in the file
cabinet located in the section and in the Quality Manual.
3.12 Quality Assurance Manual
This manual, in its entirety, along with the support manuals,
represents the documented QA manual.
3.13 Equipment Inventory
Specific requirements regarding equipment inventory are included in
section 5.1 of this manual. The equipment inventory is also kept on a
computer database and in the green file cabinet in the Serology
Section.
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3.14 Safety Manual
The Safety Officer maintains documentation regarding safety
procedures. The DNA analysis section complies with the requirements
of the Laboratory Safety Manual.
3.15 Material Safety Data Sheets (MSDS)
The MSDS are located in the hallway of the laboratory near the
catalogues for supplies and also on CD ROM.
3.16 Historical or Archival Records
Archived records are filed in the Serology section.
3.17 License and Certificates
Analysts will provide the Laboratory Director with copies of training
certificates for inclusion in their personnel files. Analysts also keep
copies of certificates obtained.
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4. Validation
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
4.1 General Considerations for Developmental Validation of the DNA
Analysis Procedure
4.1.1 The following is a list of validation requirements and standards that
must be considered prior to the forensic implementation of any DNA
procedure or locus. The validation process provides the information
necessary to assess the ability of a procedure to reliably obtain a desired
result, determines the conditions under which such results can be obtained,
determines the limitations of the procedure, and identifies the critical aspects
of the procedure that must be carefully controlled and monitored.
4.1.2 Validation for the PCR STR procedure in use in the Hamilton County
Coroner’s Office has been completed and is documented in official laboratory
manuals and/or in the appropriate scientific literature, as noted for each
specific validation area.
4.1.3 Each new locus will be validated with appropriate studies of limited
scope (e.g., non-probative casework, sensitivity studies, mixture studies).
4.1.4 The FBI 13 core loci were selected for use by the Hamilton County
Coroner’s Office and are readily available to the forensic science community.
4.1.5 The validation process should include the following studies:
4.1.5.1 Consistency
Using specimens supplied by proficiency test vendors and by other
laboratories, the reproducibility of the technique has been evaluated
both within the laboratory and among different laboratories.
These results are located in a separate validation manual, and in the
scientific literature:
AmpFiSTR Profiler Plus Amplification Users Manual.
10
Wallin et al., “TWGDAM Validation of AmpFiSTR Blue PCR Kit for for
Forensic Casework Analysis”, J. For. Sci., Vol. 43, 1998, pp. 854-870.
4.1.5.2 Mixed Specimen Studies
The system has been investigated to determine its ability to detect the
components of a mixed specimen and to define its limitations.
This system is also documented in a separate Validation Manual, and
in the scientific literature:
AmpFiSTR Profiler Plus Amplification Users Manual.
Wallin et al., “TWGDAM Validation of AmpFiSTR Blue PCR Kit for for
Forensic Casework Analysis”, J. For. Sci., Vol. 43, 1998, pp. 854-870.
4.1.5.3 Nonprobative Evidence
DNA types have been examined in non-probative evidentiary stained
materials.
These results are documented in a separate Validation Manual, and in
the scientific literature:
AmpFiSTR Profiler Plus Amplification Users Manual.
Wallin et al., “TWGDAM Validation of AmpFiSTR Blue PCR Kit for for
Forensic Casework Analysis”, J. For. Sci., Vol. 43, 1998, pp. 854-870.
11
4.1.5.4 Percent Stutter Study
Stutter bands are minor product bands which differ from the main
allele band by 4 base pairs. The amount of stutter product increases
with increased repeat number for an allele at a locus. It is helpful to
quantitate the stutter product present in known single contributor
samples.
This is documented in a separate Validation Manual, in the scientific
literature:
AmpFiSTR Profiler Plus Amplification Users Manual.
Gill et al., “Development of Guidelines to Designate Alleles Using an
STR Multiplex System”, For. Sci. Intl., Vol. 89, 1997, pp. 185-197.
Walsh et al., “Sequence Analysis and Characterization of Stutter
Products at the Tetranucleotide Repeat Locus vWA”, Nucleic Acids
Res., Vol. 24 , 1996, pp. 2807-2812.
4.1.5.5 Sensitivity Study
The maximum and minimum amount of sample to give reliable results
has been determined and is documented in a separate Validation
Manual, and in the scientific literature:
AmpFiSTR Profiler Plus Amplification Users Manual.
Wallin et al., “TWGDAM Validation of AmpFiSTR Blue PCR Kit for for
Forensic Casework Analysis”, J. For. Sci., Vol. 43, 1998, pp. 854-870.
4.1.5.6 Peak Height Ratios
The peak heights obtained for the alleles of a heterozygote are similar
but not often identical. The peak height difference between two peaks
can be significant. The quantitation of normal peak height ratios in
known single contributor samples has been documented in a separate
Validation Manual, and in the scientific literature:
AmpFiSTR Profiler Plus Amplification Users Manual.
Wallin et al., “TWGDAM Validation of AmpFiSTR Blue PCR Kit for for
Forensic Casework Analysis”, J. For. Sci., Vol. 43, 1998, pp. 854-870.
12
4.1.5.7 Precision Study
The precision of the instrument used in DNA analysis must be tested
to determine the precision of the unit. The precision of the Hamilton
County 310 Genetic Analyzer has been documented in a separate
Validation Manual. The precision of other instruments used in other
laboratories has also been documented in the scientific literature:
AmpFiSTR Profiler Plus Amplification Users Manual.
Wallin et al., “TWGDAM Validation of AmpFiSTR Blue PCR Kit for for
Forensic Casework Analysis”, J. For. Sci., Vol. 43, 1998, pp. 854-870.
Lazaruk et al., “Genotyping of Forensic Short Tandem Repeat (STR)
Systems based on Sizing Precision in a Capillary Electrophoresis
Instrument”, Electrophoresis, Vol. 19, 1998, pp. 86-93.
The following are general references; other references can be applicable:
PCR Technology, Principles and Applications for DNA Amplification, ed.
Erlich, H. A., (1989).
AmpFiSTR Profiler Plus Amplification Users Manual.
4.2 Characterization of Loci
During the development of a DNA analysis system, basic characteristics of
the loci must be determined and documented.
4.2.1 Inheritance
“DNA loci used in forensic testing shall have been validated by family
studies to demonstrate the mode of inheritance.”
AmpFiSTR Profiler Plus Amplification Users Manual, page 12-11.
4.2.2 Gene Mapping
“The chromosomal location of the polymorphism loci used for forensic
testing shall be submitted to or recorded in the Yale Gene Library or
the International Human Gene Mapping Workshop.”
AmpFiSTR Profiler Plus Amplification Users Manual, page 12-12.
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4.2.3 Detection
“The molecular basis for detecting the polymorphic loci used for
forensic testing shall be documented in the scientific or technical
literature.”
AmpFiSTR Profiler Plus Amplification Users Manual, page 12-12.
4.2.4 Polymorphism
“The type of polymorphism shall be known”
AmpFiSTR Profiler Plus Amplification Users Manual, page 12-13.
****Section 4.3 deals with RFLP Procedures only.
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4.4 Specific Developmental Validation of PCR Based DNA Procedures
4.4.1 Amplification
4.4.1.1 The PCR primers must be of known sequence
AmpFiSTR Profiler Plus Amplification Users Manual, page 1214.
4.1.1.2 Protection
Conditions and measures have been established to protect preamplified samples from contamination with post PCR materials
as follows:
a. There is an extraction area for recovery of DNA samples.
b. A separate Pre-PCR area is available for set-up of preamplified samples.
c. A PCR area is available for the amplification, development
and storage of amplified product.
AmpFiSTR Profiler Plus Amplification Users Manual, page 1214.
4.4.1.3 Conditions
The reaction conditions such as thermocycling parameters and
critical reagent concentrations (primers, polymerase and salts)
have been determined to provide the required specificity.
AmpFiSTR Profiler Plus Amplification Users Manual, page 1215.
4.4.1.4 Cycles
The number of cycles required to produce reliable results has
been determined.
AmpFiSTR Profiler Plus Amplification Users Manual, page 1216.
4.4.1.5 Differential Amplification
15
Potential for differential amplification has been addressed and
is documented in scientific literature.
AmpFiSTR Profiler Plus Amplification Users Manual, page 1216 through 12-18.
4.5 Internal Validation of Established Procedures
Prior to implementing a new procedure, a substantially modified existing
procedure, or an existing DNA procedure developed by another laboratory, inhouse validity and reliability of the procedure must be demonstrated and
documented. This internal validation must included the following:
4.5.1 The method must be tested using known samples.
-Samples from lab members were analyzed. Including different
body tissues, and different extraction techniques.
Documentation for tests is located in the STR Validation
manuals and also on Jazz discs in the PCR amplification area.
-Samples from expired CTS proficiency tests were analyzed.
Documentation for tests is located in the STR Consistency
manual and also on Jazz discs in the PCR amplification area.
-Samples received from the Rhode Island Department of Health
were analyzed. Documentation for tests is located in the STR
Consistency manual and also on Jazz discs in the PCR
amplification area.
4.5.2 If a modification which materially effects the results of an
analysis has been made to an analytical procedure, the modified
procedure must be compared to the original using identical samples.
4.5.3 Precision must be determined by repetitive analyses to establish
criteria for matching. Documentation for tests is located in the STR
Precision manual and on Jazz discs in the PCR amplification area.
4.5.4 The laboratory must demonstrate that its procedures do not
introduce contamination which would lead to errors in typing.
-Proper use of controls protects against contamination and
errors in typing.
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-Ongoing proficiency testing protects against contamination and
errors in typing.
4.5.5 The method must be tested using proficiency test samples. The
proficiency test may be administered internally, externally, or
collaboratively.
-Ongoing proficiency testing takes place in the section.
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5. Equipment, Materials and Facilities
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
5.1 Equipment
Only suitable and properly operating equipment can be employed. To
ensure this, critical parameters are monitored and documented to
maintain successful operation of the typing technique.
5.1.1 A list of equipment used will be maintained to include (if
possible) name of item, manufacturer, Model, serial number, and
acquisition date. This list is kept in the green file cabinet in the
section.
5.1.2 Manufacturer’s operation manuals are available in the Serology
Section.
5.1.3 Routine maintenance and calibration logs will be kept for all
instruments and equipment used in the section. These logs are kept in
the Calibration and Maintenance Logbook for the Serology section.
5.1.4 Instruments and equipment that are dedicated to a particular
area will be marked as dedicated.
5.2 Materials and Reagents
Chemicals and reagents should be of suitable quality, correctly
prepared, and demonstrated to be compatible with the methods
employed.
5.2.1 Logs are maintained of commercial supplies and kits which
have expiration dates.
5.2.2 When reagents are made, they are to be logged into the chemical
quality control manual including formulation of reagent, lot numbers
of chemicals used, concentration, date prepared, and person making
the reagent. A lot number is to be assigned and noted on the reagent
bottle.
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5.2.3 When reagents are made, they should be labeled properly,
including information on identity, concentration, date of preparation,
identity of individual preparing reagents, special storage requirements
and expiration date, where appropriate.
5.2.4 A current inventory of supplies and materials is maintained and
includes information on supplier, catalog number, lot number, date
received, dated opened, date ordered, and cost.
5.2.5 Dedicated materials and reagents are marked as dedicated.
5.2.6 Specific procedures for cleaning, preparing and sterilizing
glassware and plastic supplies are located in the Calibration and
Maintenance Logbook.
5.3 Laboratory Facilities for PCR Analysis
The PCR laboratory will require special laboratory configuration and sample
handling.
5.3.1 Examination work area - All preliminary sample collection and
preparation will be done in the peninsular table area of the Serology
Section, the Trace Evidence Laboratory or the vehicle examination
garage.
5.3.2 Extraction work area - This work area is for sample extraction
and concentration. It is physically separate from the amplified DNA
work area and the PCR set-up area to eliminate the possibility of
cross-contamination.
5.3.3 PCR set-up work area - This area is isolated from the extraction
area to ensure that the reaction mix cocktails are prepared in a clean
environment. All PCR set-up will be done in the biological safety
cabinet. This biological safety cabinet is to be used only for PCR set-up
and preparation of reaction mix cocktails.
5.3.4 Amplified DNA work area - This work area is physically
separate, within the laboratory, for containment of amplified DNA
product. This area includes the amplification area with the
thermalcycler, and space for all procedures utilizing the product for
typing. All equipment and reagents used in this area are dedicated to
this area and are not to be used in either the extraction or PCR set-up
areas. Amplified DNA should be stored and disposed of in this area.
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5.3.5 Decontamination - The decontamination procedure for cleaning
and decontamination of facilities and equipment from DNA and PCR
product DNA is contained in the Calibration and Maintenance Manual
for the Serology section.
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6. Evidence Handling
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: Version 1-1-2000
Reviewed by: Quality Manager
Reviewed: Annually
Revised: December 24, 2002
Evidence items are collected, received, handled, sampled and stored so as to
preserve the identity, integrity, condition and security of the item.
6.1 Sample Labeling
Each item of evidence should be labeled with a “CL” number, a Q- or Knumber, and the analyst’s initials. The Q- (Questioned) and K- (Known) item
numbers will be assigned according to the Laboratory’s Administrative
procedures.
6.2 Chain of Custody
A clear well documented chain of custody must be maintained from the time
the evidence is first received until it is released from the laboratory.
6.2.1 Sealing of Evidence
During analysis of evidence, the analyst should avoid damaging the
seals on evidence made by others. When possible, evidence should be
cut open in an area not covered by evidence tape.
Following analysis, the analyst should seal any openings made in the
packaging with evidence tape. The analyst should mark the evidence
tape with his/her initials and date of seal.
6.2.2 Acceptance of Evidence
Evidence submitted to the laboratory shall be accepted by an Evidence
Technician or a serology analyst when necessary (i.e. fetal tissue etc.).
The evidence should be marked as received by the Evidence Technician
with the initials and date of receipt. Blood and urine tubes may be
included inside of or attached to sexual assault kits for the purpose of
toxicological testing. Upon receipt of a sexual assault kit containing
blood and/or urine tubes, the kit should be cut open and these tubes
removed. The tubes are then logged in and marked with the bar code
label, and transferred to the toxicology refrigerator. The sexual
assault kit is then resealed with evidence tape and the kit marked
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with the Evidence Technician’s initials and the date the tubes were
removed.
Incoming serology evidence is checked against the submission sheet to
ensure that all items of evidence have been included on the submission
sheet. The submission sheet is initialed and dated in the “receipt of
evidence” area. After the case has been logged in by the Evidence
Technician, and the barcode affixed, the evidence is transferred to the
serology store room.
6.2.3 Release of Evidence
Evidence will only be released to the submitting agency, or the
prosecutor’s office. Any release of evidence is documented on the back
of the submission sheet. The Evidence Technician releasing the
evidence must initial and date the submission sheet in the area of
“released evidence”.
6.3 Sample Handling and Storage
The following policy ensures that evidence sampled will be handled,
processed and preserved so as to protect against loss, contamination or
deleterious change. Whenever possible, a portion of the original sample
submission is retained or returned to the submitting agency.
6.3.1 Acceptance for DNA Analysis
The criteria associated with accepting samples for DNA testing will be
based on the following:
A. Quantity of Sample.
B. Presence of Seminal Fluid in sexual assault cases.
C. Availability of blood standards or buccal swab samples from the
individuals involved (or believed to be involved) in the case.
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6.3.2 Additional Samples
In cases where additional samples are needed, an analyst will
coordinate and document the procurement of these samples with the
detective and or the prosecutor in the case.
6.3.3 Sample Labeling
Each working sample must be labeled with a unique identifier. This
consists of the case number, analyst’s initials, date, and individual
item number such as “Q”, “K”, or name.
6.3.4 Storage
A. Short term storage
Upon receipt, all biological evidence is stored at room temperature in
the locked evidence lockers or in the locked file cabinet in the Serology
section. Blood and urine tubes are stored in locked refrigerators in the
toxicology or serology sections.
B. Long term storage
Upon completion of the DNA testing, the DNA analyst has the
ultimate responsibility for long term storage of the refrigerated/frozen
samples. A portion of the sample that was tested as well as the DNA
extract will be retained in the DNA freezer.
Buccal swab specimens collected by police agencies will be returned to
the submitting agency. DNA extracts of those samples from
individuals who have been excluded from the case in question will be
destroyed by the DNA analyst at the Hamilton County Coroner’s
Laboratory. This policy will be applied retroactively.
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7. Analytical Procedure
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised: December 24, 2002
7.1 Sample Evaluation and Preparation
7.1.1 General characterization of the biological material will be
performed prior to DNA analysis. Evidentiary samples submitted will
be evaluated to determine the appropriateness for DNA
analysis.
7.1.2 When semen is identified, a method of differential extraction will
be employed.
7.1.3 Testing of evidence and evidentiary samples will be conducted to
provide the maximum information with the least consumption of the
sample. Whenever possible, a portion of the original sample will be
retained or returned to the submitting agency as established by
laboratory policy.
7.2 PCR Standards and Controls
At each step of the testing procedure, standards and controls must be applied
that not only evaluate the effectiveness of the testing process but also ensure
that the procedure is being properly performed.
7.2.1 DNA Isolation
Sample contamination
The DNA isolation procedure protects against sample
cross contamination through a witnessing system in
which another analyst witnesses any transferring
procedures.
The DNA extraction of evidence samples is performed at a
separate time from the DNA extraction of reference
samples. This precaution will help to prevent potential
cross contamination between evidence samples and
reference samples.
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7.2.2 Effectiveness
The DNA isolation quantitation and typing procedure is
evaluated by periodic use of an appropriate source of
human DNA. The internal proficiency test described in
the Training Manual for the Serology section utilizes
known blood samples which will be extracted, amplified
and typed. A NIST standard or NIST traceable standard
will also be tested once yearly and the results will be
documented in the Critical Reagents logbook.
Reagent Blank
With each set of extractions a reagent blank must be
used. The reagent blank consists of only the reagents
used in the test process, must include all reagents, and is
processed through the entire typing procedure alongside
the evidence samples. If more than one type of extraction
procedure is used (with different extraction reagents),
then a reagent blank should be set up for each type of
extraction or group of extraction reagents used.
7.3 DNA Recovery
To estimate the quantity of DNA recovered from the specimens,
a slot blot will be performed. An appropriate set of human DNA
standards will be used with the slot blot procedure.
****Section 7.4 deals with RFLP analysis only.
7.5 Analytical Procedures for PCR-Based Techniques
7.5.1 Internal Controls and Standards
7.5.1.1 Negative controls to be included with each sample set
are:
(a) A reagent blank
(b) An amplification blank (negative control)
7.5.1.2 A human DNA known type supplied by the
manufacturer is amplified as a positive control and carried
through the remainder of the typing.
25
7.5.1.3 Substrate controls may be collected from the evidence
(e.g. unstained areas adjacent to stained areas, hair shafts
adjacent to hair roots) and may be processed at the same time as
the evidence samples.
7.5.1.4 Where feasible, the sample will be split for duplicate
analysis as early as possible prior to amplification.
26
Polymerase Chain Reaction (PCR)
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Short Tandem Repeat (STR) genetic markers are polymorphic DNA
loci that contain a repeated nucleotide sequence. The number of repeat units
at an STR locus differs from individual to individual, so alleles of many
different lengths are possible. The STR loci can be amplified using the
polymerase chain reaction process(PCR).
PCR is an enzymatic process in which a specific region of DNA is
replicated to produce several million copies of that particular sequence. The
method by which the PCR reaction amplifies the DNA follows three steps:
1. First the two strands of DNA are separated by denaturing the DNA
with heat.
2. Next, short pieces of laboratory synthesized DNA, called primers,
are hybridized to each DNA strand by lowering the temperature of the
system. The primers flank the target sequence of the DNA strand
which is polymorphic.
3. Finally, the primers are extended by the enzyme Taq DNA
polymerase. The DNA polymerase will start at the end of each of
the two strands and links nucleotides in the precise order specified
by the “template” DNA strands.
The cyclic repetition of these steps results in the doubling of the
amount of DNA that was present at the beginning of the cycle. At the end of
approximately 20-25 cycles, enough product should have been produced to
detect the different alleles associated with the different loci being examined.
Following amplification, a portion of the amplified DNA is separated
by capillary electrophoresis and detected by laser excitation of fluorescent
tags. These “excited” fluorescent tags emit a higher wavelength which is
collected on a CCD camera. The alleles appear as peaks on
electropherograms.
Extraction Special Precautions
27
1. All extraction steps and procedures must be performed in the extraction
work area.
2. Prepare extracts of known samples and questioned samples at different
times. This will help prevent potential cross-contamination between
evidence samples and reference samples.
3. Use reagents and equipment dedicated to the operations of extraction.
4. Perform DNA extraction from samples containing high levels of DNA (ex.
whole blood) separately from samples containing a low level of DNA (ex.
single hairs) to minimize the potential for sample to sample contamination.
5. Use a clean cutting surface (weighing paper) for each piece of evidence.
6. Clean scissors and tweezers thoroughly with 10% bleach and water after
cutting each evidence sample.
7. Use disposable gloves at all times and change gloves frequently to avoid
sample to sample contamination.
8. Use disposable pipette tips and microcentrifuge tubes.
9. Always change pipette tips between handling each sample.
10. Store reagents as small aliquots to minimize the number of times a given
tube of reagent is opened.
11. Avoid splashes by centrifuging all liquids to the bottom of the tubes
before opening. Use a microtube decapping device to open closed tubes.
12. Include a reagent blank control with each set of DNA extractions to
check for the presence of contaminating DNA in the reagents.
13. Never “blow out” the last bit of sample from a pipettor.
14. Limit the number of samples handled in a single run to a manageable
number.
28
Special Precautions for PCR Set Up
1. Use dedicated pipets for preparing the master PCR Reaction Mix and for
adding sample DNA to the PCR Reaction Mix. Keep these pipets in the PCR
set up area. Never “blow out” the last bit of sample from the pipet.
2. Always add DNA to the PCR Reaction Mix last. This minimizes crosscontamination by reducing the number of opportunities for inadvertent
transfer of DNA between samples.
3. After the addition of each DNA sample, cap the tube before proceeding to
the next sample.
4. Cap the negative control tube last, after all DNA samples have been added
to the other tubes. This control will provide as a check for contamination
occurring during PCR setup.
5. Avoid touching the inside surface of the tube caps.
6. Change pipet tips after addition of each sample DNA to a PCR Reaction
Mix.
7. Store the DNA Amplification Reagents together in the box provided to
serve as a barrier to possible contamination.
29
Special Precautions for Amplified DNA
1. Always remove gloves when leaving the Amplified DNA Work Area to
avoid the transfer of amplified DNA into other work areas.
2. Reduce the unnecessary dispersal of DNA around the work area by
changing gloves whenever they may have become contaminated with
amplified DNA.
3. Avoid splashing by opening tubes containing amplified DNA carefully. A
microtube de-capping device makes it easier to open the tubes.
4. Use disposable bench paper to cover the work area used to perform the
typing steps to prevent the accumulation of amplified DNA on permanent
work surfaces. Diluted bleach should be used periodically to wash exposed
work areas.
5. Use the Thermal Cycler only for amplification and denaturation of
amplified DNA for typing. Never use the Thermal Cycler for incubation of
tubes containing unamplified DNA.
6. Store tubes of amplified DNA in the amplified DNA work area.
30
Extraction of DNA from Non-semen Body Fluid Stains or
Whole Blood Using Chelex
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Caution: All extraction steps must be performed in the DNA
extraction area. Extract known samples at a different
time than that of the questioned samples.
Principle:
The chelex extraction procedure for use on samples is a
rapid and simple method for extracting DNA.
Materials:
Phosphate Buffered Saline Solution
5% Chelex Solution
1.5 milliliter microcentrifuge tubes
Procedure:
1. For liquid blood, add up to 50 microliters to a microcentrifuge
tube. For stains, cut the stain into small pieces and place in a
microcentrifuge tube. Wipe scissors and forceps with bleach
followed by deionized water.
This step must be witnessed.
Pipette 1 milliliter of sterile PBS into the tube and vortex
for 2 seconds.
2. Incubate at room temperature for 30 minutes and mix
occasionally by inversion.
3. Spin in a microcentrifuge for 2 - 3 minutes at maximum
speed to spin down the white blood cells.
4. Without disturbing the cell pellet, carefully remove and
discard all but about 20 to 30 microliters of the supernatant. If
the sample is a bloodstain, leave the fabric substrate in the tube
with the cell pellet.
5. Add 200 microliters of 5% Chelex to each tube. Vortex tube
for 10 seconds.
31
6. Incubate at 56oC for 30 minutes.
7. Vortex tube for 10 seconds.
8. Incubate tube in a boiling water bath for 8 minutes.
9. Vortex tube for 10 seconds.
10. Spin in a microcentrifuge for 3 minutes at maximum speed.
11. The supernatant contains the DNA and is now ready for
quantitation by slot blot hybridization.
12. The sample is now ready for amplification.
13. Store samples at 4oC or frozen. Prior to reuse of
these samples for amplification, repeat steps 9 and 10.
Critical Aspects
& Limitations:
Chelex beads must be evenly distributed in solution while
pipetting. Pipette the volume needed for each sample
directly from the beaker after the beads have been mixed
into the solution.
32
Extraction of DNA from Oral Swabs Using Chelex
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Caution: All extraction steps must be performed in the DNA
extraction area. Extract known samples at a different
time than that of the questioned samples.
Principle:
The chelex extraction procedure for use on samples is a
rapid and simple method for extracting DNA.
Materials:
1.5 milliliter microcentrifuge tubes
5% Chelex Solution
Procedure:
1. Cut the stain into small pieces and place in a microcentrifuge
tube. Wipe scissors and forceps with bleach followed by
deionized water.
This step must be witnessed.
2. Add 200 microliters of 5% Chelex Solution to the sample.
3. Vortex the tube for 10 seconds.
4. Incubate the tube at 56o for 30 minutes.
5. Vortex the tube for 10 seconds.
6. Incubate in a boiling water bath for 8 minutes.
7. Vortex the tube for 10 seconds.
8. Spin in a microcentrifuge for 3 minutes at maximum speed.
9. Estimate the amount of DNA by slot blot hybridization.
10. After quantitation, the sample can be amplified.
33
11. Store samples at 4oC or frozen. Prior to use of samples after
storage, they should be vortexed briefly and spun in a
microcentrifuge for 5 seconds. (quick spin)
Critical Aspects
& Limitations:
Chelex beads must be evenly distributed in solution while
pipetting. Pipette the volume needed for each sample
directly from the beaker after the beads have been mixed
into the solution.
34
Extraction of DNA from Hair Using Chelex
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Caution: All extraction steps must be performed in the DNA
extraction area. Extract known samples at a different
time than that of the questioned samples.
Principle:
The chelex extraction procedure for use on samples is a
rapid and simple method for extracting DNA.
Materials:
1.5 milliliter microcentrifuge tubes
5% Chelex Solution
Proteinase K
Procedure:
I. Unmounted Hairs
1. Rinse the hair thoroughly in 100% ethanol. Follow the
ethanol rinse with a thorough rinse in sterile deionized water.
2. Place at least 1 centimeter of the hair root end into a 1.5
milliliter microcentrifuge tube. Wipe scissors and forceps with
bleach followed by deionized water.
This step must be witnessed.
3. Add 200 microliters of 5% Chelex 100 solution into the
microcentrifuge tube. Add 2 microliters of proteinase K. Make
sure that the hair is submerged in the solution.
4. Incubate the samples at 56oC at least six to eight hours or
overnight.
5. Vortex at high speed for 5 to 10 seconds.
6. Quick spin in a microcentrifuge for 5 seconds.
35
7. Check that the hair is completely immersed in the Chelex
100 solution and incubate the samples in a boiling water bath
for 8 minutes.
8. Vortex the samples for 10 seconds.
9. Spin in a microcentrifuge for 3 minutes.
10. Estimate the amount of DNA in the samples by slot blot
hybridization.
11. After quantitation the samples can be amplified.
12. Store samples at 4oC or frozen. Prior to reuse of these
samples for amplification repeat steps 8 and 9.
II. Slide-Mounted Hair Specimens
1. Loosen the slide coverslip by carefully pipetting xylene
around the coverslip edges. If the coverslip will not loosen, the
entire slide can be soaked in xylene for one or more hours until
the coverslip has loosened.
2. After removal of the coverslip, remove the hair and rinse it
thoroughly with xylene.
3. Continue processing at step one of the procedure for
unmounted hair specimens.
Critical Aspects
& Limitations:
Chelex beads must be evenly distributed in solution while
pipetting. Pipette the volume needed for each sample
directly from the beaker after the beads have been mixed
into the solution.
36
Extraction of DNA from Non-semen Body Fluid Stains or
Whole Blood Using Organic Methods
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Caution: All extraction steps must be performed in the DNA
extraction area. Extract known samples at a different
time than that of the questioned samples.
Principle:
The organic extraction procedure utilizes a
phenol/chloroform solution which removes proteins from
the DNA by denaturing them.
Materials:
Stain Extraction Buffer
Proteinase K
TE Buffer
Phenol/Chloroform/Isoamyl Alcohol
Microcon Concentrator
Spin Basket tubes
Procedure:
1. Liquid blood samples should be made into bloodstains. Cut
the stain into small pieces and place in a spin ease tube. Wipe
scissors and forceps with bleach followed by deionized water.
Cigarette Butts: Remove the paper from the filter end of the
cigarette butt and cut into smaller pieces.
Chewing Gum: Cut the gum into smaller pieces. Prior
refrigeration may aid the process.
This step must be witnessed.
2. To the sample add 300 microliters of stain extraction buffer
and 7.5 microliters of proteinase K solution. Vortex for 1 second
and spin in a microcentrifuge for 2 seconds (quick spin) to force
the cutting into the extraction fluid.
37
3. Incubate the tube at 56oC overnight. (18 hours
minimum/24 hours maximum)
4. Spin in a microcentrifuge for 2 seconds (quick spin) to force
condensate into the bottom of the tube.
5. Using a wooden applicator stick, transfer the cutting into a
spin basket and place the basket into the microcentrifuge tube
containing the stain extract. Spin in a microcentrifuge for 5
minutes.
6. Remove and discard the basket insert and cutting into a
biohazard container.
7. In a fume hood, add 300 microliters of
phenol/chloroform/isoamyl alcohol to the stain extract. Vortex
the mixture to attain a milky emmulsion. Spin the tube in a
microcentrifuge for 3 minutes to separate the 2 phases.
8. To a Microcon concentrator add 100 microliters of TE buffer.
Transfer the aqueous phase (the upper layer) from the tube in
step 7 to the concentrator. Avoid pipetting organic solvent from
the tube into the concentrator.
This step must be witnessed.
9. Place a spin cap on the concentrator and spin in a
microcentrifuge at 500 x g for 10 minutes. The DNA sample will
remain concentrated in about 20 - 40 microliters of TE buffer in
the bottom of the upper microcon reservoir and molecules with a
molecular weight of less than about 100,000 daltons will pass
through the filter.
10. Carefully remove the concentrator unit from the assembly
and discard the fluid from the filtrate cup. Return the
concentrator to the top of the filtrate cup.
11. Remove the spin cap and add 200 microliters of TE to the
concentrator. Replace the spin cap and spin the assembly in a
microcentrifuge at 500 x g for 10 minutes.
12. Remove the spin cap and add a measured volume of TE that
is between 40 microliters and 200 microliters to the
38
concentrator. Remove the concentrator from the filtrate cup and
carefully invert the concentrator onto a labeled retentate cup.
Discard the filtrate cup.
13. Spin the assembly in a microcentrifuge at 500 x g for 5
minutes to transfer the DNA concentrate into the cup.
14. Discard the concentrator. Cap the retentate cup containing
the DNA sample.
15. Estimate the quantity of DNA in the sample by slot blot
hybridization.
16. After quantification the sample can be amplified.
17. Store samples at 4oC or frozen. Prior to the use of samples
after storage they should be vortexed briefly and spun
in a
microcentrifuge for 5 seconds (quick spin).
Critical Aspects
& Limitations:
DNA extracted from bloodstain evidence is sometimes
resistant to amplification by PCR. This resistance may be
due to red blood cell components including heme
compounds inhibiting PCR. Discoloration of the DNA
extract (retentate) is usually predictive of inhibition. To
overcome this potential inhibition, the DNA extract can
be subjected to several more TE washes using the
microcon.
39
Extraction of DNA from Semen Containing Stains Using
Organic Methods
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Caution: All extraction steps must be performed in the DNA
extraction area. Extract known samples at a different
time than that of the questioned samples.
Principle:
The organic extraction procedure utilizes a
phenol/chloroform solution which removes proteins from
the DNA by denaturing them. This procedure also
utilizes a differential extraction to separate the sperm
fraction (male) from the female fraction.
Materials:
Proteinase K
20% Sarkosyl Solution
DTT
Phenol/Chloroform/Isoamyl Alcohol
TNE Buffer
Sperm Wash Buffer
TE Buffer
Spin Basket Tubes
Microcon Concentrator
Procedure:
1. Cut swab or fabric using scissors. Use a fresh cutting surface
for each sample tested. Add the swab or fabric cutting to a Spinease tube. Wipe scissors and forceps with bleach followed by
deionized water.
This step must be witnessed.
2. To the sample add 400 microliters of TNE buffer, 25
microliters of 20% Sarkosyl solution, 75 microliters deionized
water and 5 microliters of proteinase K. Vortex for 1 second and
spin in a microcentrifuge for 2 seconds (quick spin) to force the
material into the extraction fluid.
40
3. Incubate at 37oC for 2 hours to lyse the epithelial
cells.
4. Using a wooden applicator stick, transfer the swab or fabric
cutting into a spin basket and place the spin basket into the
tube containing the stain extract. Spin in a microcentrifuge at
maximum speed for 5 minutes.
5. Remove the basket insert from the extract tube. Remove the
swab material from the basket, place in a clean microcentrifuge
tube and store frozen.
6. Remove the supernatant fluid from the extract being careful
not to disturb any pelleted material. Place the supernatant into
a new labeled tube. This supernatant is the female
fraction. Analysis of the female fraction resumes at step
11. The pellet remaining in the tube is the cell pellet.
This step must be witnessed.
7. Wash the cell pellet by resuspending it in 1000 microliters of
sperm wash buffer, vortexing the suspension briefly, and
spinning the tube in a microcentrifuge at maximum speed for 5
minutes. Remove and discard the supernatant fluid, being
careful not to disturb the cell pellet.
8. Repeat step 7 two additional times for a total of three washes
of the cell pellet.
9. To the tube containing the washed pellet, add 150 microliters
TNE buffer, 50 microliters of 20% Sarkosyl solution, 150
microliters of deionized water 7 microliters of DTT, and 10
microliters of proteinase K. Close the tube caps and vortex for 1
second and spin in a microcentrifuge for 2 seconds (quick spin)
to force all fluid and material to the bottoms of the tubes.
10. Incubate at 37o for 2 hours.
11. To the tube containing the cell pellet and to the tube
containing the female fraction, add 400 microliters of
phenol/chloroform/isoamyl alcohol. Vortex the mixture to attain
a milky emulsion. Spin the tube in a microcentrifuge for 3
minutes.
41
12. Assemble a microcon unit. To the top of the
concentrator, add 100 microliters of TE buffer. Transfer the
aqueous phase (upper layer) from the tube in step 11 to the top
of the concentrator. Avoid pipetting organic solvent from the
tube into the concentrator.
This step must be witnessed.
13. Place a spin cap on the concentrator and spin in a
microcentrifuge at 500 X g for 10 minutes.
14. Carefully remove the concentrator unit from the assembly
and discard the filtrate fluid from the filtrate cup. Return the
concentrator to the top of the filtrate cup.
15. Remove the spin cap and add 200 microliters of TE buffer to
the concentrator. Replace the spin cap and spin the assembly
in a microcentrifuge at 500 X g for 10 minutes.
16. Remove the spin cap and add a measured volume of TE that
is between 40 microliters and 200 microliters to the
concentrator. Remove the concentrator from the filtrate cup and
carefully invert the concentrator onto a labeled retentate cup.
Discard the filtrate cup.
17. Spin the assembly in a microcentrifuge at 500 X g for 5
minutes.
18. Discard the concentrator. Cap the retentate cup.
19. Estimate the quantity of DNA in the sample by slot blot
hybridization.
20. After quantification, the sample can be amplified.
21. Store samples at 4oC or frozen. Prior to the use of samples
after storage they should be vortexed briefly and spun
in a
microcentrifuge for 5 seconds (quick spin).
42
Extraction of DNA from Hairs Using Organic Methods
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Caution: All extraction steps must be performed in the DNA
extraction area. Extract known samples at a different
time than that of the questioned samples.
Principle:
The organic extraction procedure utilizes a
phenol/chloroform solution, which removes proteins from
the DNA by denaturing them.
Materials:
Stain Extraction Buffer
Proteinase K
TE Buffer
DTT
Phenol/Chloroform/Isoamyl Alcohol
Microcon Concentrator
Spin Basket tubes
Procedure:
Unmounted Hairs
1. Rinse the hair thoroughly in sterile distilled water by
shaking for 1 hour.
2. Rinse hair briefly in fresh sterile deionized water.
3. Place at least 1 centimeter of the hair root end into a spinease tube. Wipe scissors and forceps with bleach followed by
deionized water.
This step must be witnessed.
4. Add 500 microliters of stain extraction buffer, 50 microliters
of DTT and 15 microliters of proteinase K solution to the
sample. Vortex for 1 second and quick spin in a microcentrifuge
to force the hair into the extraction fluid.
43
5. Incubate the tube at 56C overnight.
6. Quick spin in a microcentrifuge to force the condensate into
the bottom of the tube.
7. Add an additional 50 microliter of DTT and 15 microliters of
Proteinase K, and incubate at 56oC overnight.
8. In a fume hood, add 300 microliters of
phenol/chloroform/isoamyl alcohol to the extract. Vortex the
mixture briefly to attain a milky emulsion. Spin the tube in a
microcentrifuge for 3 minutes.
9. Add 100 microliters of TE Buffer to a Microcon Concentrator.
Transfer the aqueous phase from the tube in step 6 to the
concentrator. Avoid pipetting organic solvent from the tube into
the concentrator.
This step must be witnessed.
10. Place a spin cap on the concentrator and spin in a
microcentrifuge at 500 X g (3000 rpm) for 10 minutes.
11. Remove the concentrator unit from the assembly and
discard the fluid from the filtrate cup. Return the concentrator
to the top of the filtrate cup.
12. Remove the spin cap and add 200 microliters of TE Buffer to
the concentrator. Replace the spin cap and spin the assembly in
a microcentrifuge at 500 X g (3000 rpm) for 10 minutes.
13. Remove the spin cap and add 50 microliters of TE Buffer to
the concentrator. Remove the concentrator from the filtrate cup
and carefully invert the concentrator onto a labeled retentate
cup. Discard the filtrate cup.
14. Spin the assembly in a microcentrifuge at 500 X g (3000
rpm) for 5 minutes.
15. Discard the concentrator. Cap the retentate cup.
16.
Estimate the quantity of DNA in the sample by Slot Blot
hybridization. After quantification the samples can be
amplified.
44
Slide-Mounted Hair Specimens
1. Loosen the slide coverslip by carefully pipetting xylene
around the coverslip edges. If the coverslip will not loosen, the
entire slide can be soaked in xylene for one or more hours until
the coverslip has loosened.
2. After removal of the coverslip, remove the hair and rinse it
thoroughly with xylene.
3. Continue processing at step one of the procedure for
unmounted hair specimens.
45
Extraction of DNA from Bone Using Organic Methods
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Caution: All extraction steps must be performed in the DNA
extraction area. Extract known samples at a different
time than that of the questioned samples.
Principle:
The organic extraction procedure utilizes a
phenol/chloroform solution which removes proteins from
the DNA by denaturing them.
Materials:
0.5 M EDTA
Stain Extraction Buffer
Proteinase K
TE Buffer
DTT
Phenol/Chloroform/Isoamyl Alcohol
Centricon Concentrator
15 ML conical tube
The following procedure is adapted from the BCI DNA manual:
1. Clean the bone. Remove all flesh Soak the bone in 10% bleach for 1-2
minutes. Rinse and dry the bone thoroughly. Use sandpaper to remove the
outer layer of tissue. This may need to be done in a hood. Avoid sawed ends
of the bone because they may be contaminated. Avoid the marrow.
NOTE: This step is not necessary if the bone is relatively clean. It is to
protect against contamination that may occur during the autopsy.
2. Ask a pathologist to assist in generating 5g to 15 g of bone dust. Make
sure a clean stryker saw blade is used.
The following is adapted from the Journal of Forensic Sciences, Volume 36,
pp 1649-1661:
3. Place up to 1 g of bone dust in a 15 ml conical bottom tube or 3 g of bone
dust in a 50 ml conical bottom tube.
46
4. To decalcify the bone: Fill the tube nearly to the top with 0.5 M EDTA
(500mM). Rotate for 24 hours. Spin the tubes for 15 minutes at 4000 rpm
(2000g). Discard the supernatant. Refill tube with fresh 0.5 M EDTA and
repeat rotation for 5 days. It is necessary to vortex the sample at this stage
to resuspend the pellet. If one of the repetitions falls on a weekend or day off,
it is ok to let the repetition go longer. Use a tube with only EDTA as a
reagent blank.
NOTE: If using a 15 ml conical tube use the large barrel rotor on the
centrifuge and use a 50 ml conical tube as a secondary container in
case any of the tubes crack while centrifuging.
5. Fill the tube nearly to the top with distilled water, vortex to resuspend the
pellet and spin 15 minutes at 4000 rpm. Repeat two more times.
6. Resuspend the pellet in twice the volume of stain extraction buffer. Add
between 20-30 ul of proteinase K and, between 10-15 ul of DTT. Incubate
overnight at 56o C. Vortex the tubes if possible until the pellet dissolves.
7. Transfer contents from a 15 ml conical tubes to 50 ml conical tubes.
Extract 3 times with phenol/chloroform/isoamyl alcohol or until the aqueous
phase becomes relatively clear. Use parafilm to seal the tube lids. Add ~5
ML for the first extraction. Spin for 5 minutes at 2500-3000 rpm.
8. If the aqueous phase is not clear, transfer the aqueous phase to a clean 50
ml conical tube and repeat step 7. Repeat step 7 until the aqueous phase is
relatively clear. The volume of phenol/chloroform/isoamyl alcohol may be
reduced depending on the volume of each aqueous phase.
9. Transfer the aqueous phase to a Centricon 100 containing 500 ul of TE
buffer. Spin the Centricon for 10 minutes at 3000 rpm. Repeat twice.
10. Add ~200 ul of TE buffer to the Centricon filter. Invert filter over
retentate cup and spin for 2 minutes at 3000 rpm to recover the DNA into the
retentate cup.
47
11. Cover the retentate cup and place in the freezer until sample is to be
quantitated.
48
Concentrating Extracts
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
Occasionally, samples may be concentrated when the
DNA present in the sample is insufficient for typing.
Concentrating the DNA into a smaller volume can
sometimes enhance the ability to get a result from the
sample.
Procedure:
1. Add the entire volume of the DNA extract that needs
to be concentrated into a fresh microcon.
2. Spin down the sample in a microcentrifuge at 3000
rpm for approximately 10 minutes to reduce the volume of
fluid in the sample. The DNA in the sample will still be
present on the microcon filter.
3. Discard the fluid from the filtrate cup.
4. Add 20 microliters of TE buffer to the concentrator.
Remove the concentrator from the filtrate cup and
carefully invert the concentrator onto a labeled
retentate cup. Discard the filtrate cup.
This step must be witnessed.
5. Spin the assembly in a microcentrifuge at 500 X g
(3000 rpm) for 5 minutes.
6. Discard the concentrator. Cap the retentate cup.
7. Estimate the quantity of DNA in the sample by Slot
Blot hybridization. After quantification the samples can
be amplified.
49
Slot Blot Quantitation of DNA Using PE Quantiblot Kit
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
This procedure has been taken from the Perkin-Elmer Quantiblot
Human DNA Quantitation Kit product insert.
Principle:
Quantitating the amount of DNA that has been extracted
from a sample is necessary so that the optimum
concentration of DNA can be amplified. The DNA
samples, along with two-fold dilutions of DNA standards
and DNA calibrators are spotted onto a membrane
contained in a slot blot apparatus. The membrane
contains positively charged groups and binds the
negatively charged DNA. A primate-specific biotinylated
probe hybridizes with the DNA sample on the membrane.
The results are detected by chemiluminescent methods
using ECL detection reagents and an X-ray film
development procedure. The intensities of the resulting
bands are then compared with the intensities of the
standards and the concentrations are estimated.
Materials:
Quantiblot Human DNA Kit
Chemiluminescent Detection Reagents
X-ray film
Hybridization Tray
Slot Blot Apparatus
Biodyne B Membrane
Hybridization Solution
Wash Solution
Spotting Solution
Pre-wetting Solution
30% Hydrogen Peroxide
Citrate Buffer
0.5 milliliter microcentrifuge tubes
Plastic-backed paper
50
Procedure: I. Preparation of Reagents Supplied
Prepare a two-fold dilution of the DNA Standard A in TE Buffer:
1. Label 7 0.5 milliliter autoclaved microcentrifuge tubes A
through G.
2. Vortex the DNA Standard A to mix it thoroughly.
3. Transfer 120 microliters of DNA Standard A into the tube
labeled A.
4. Aliquot 60 microliters of TE Buffer into each of the six
remaining tubes labeled B through G.
5. Add 60 microliters of DNA Standard A (tube A) to the 60
microliters of TE Buffer in tube B. Vortex to mix thoroughly.
6. Add 60 microliters of diluted DNA Standard B (tube B) to the
60 microliters of TE Buffer in tube C. Vortex to mix thoroughly.
7. Add 60 microliters of diluted DNA Standard C (tube C) to the
60 microliters of TE Buffer in tube D. Vortex to mix thoroughly.
8. Continue the serial dilution through tube G.
The seven DNA standard tubes should have the concentrations of
human DNA listed below:
DNA
Standard
A
B
C
D
E
F
G
Concentration
(ng/microliter)
2
1
0.5
0.25
0.125
0.0625
0.03125
Quantity DNA per
5 microliter (ng)
10
5
2.5
1.25
0.625
0.3125
0.15625
Note: Store the diluted DNA Standards at 2o to 8oC. The DNA
Standards A through G are stable for at least three months at
2o to 8o C.
51
The Quantiblot Kit contains enough reagents for at least 10
hybridization reactions. Each hybridization reaction must include the
following 10 control samples: seven DNA Standards, the two DNA
calibrators and one blank (spotting solution only).
II. Slot Blotting
1. Determine the number of samples to be analyzed including the
seven Human DNA Standards (A through G), the DNA Calibrators 1
and 2 and the one blank (spotting solution only). Aliquot 150
microliters of spotting solution into a new 0.5 milliliter microcentrifuge
tube for each sample.
2. Label seven of the tubes containing 150 microliters of spotting
solution as follows: A, B, C, D, E, F, and G and label two of the tubes
containing 150 microliters of spotting solution as follows: DNA
Calibrator 1 and DNA Calibrator 2.
3. Vortex the seven DNA standards and the two DNA Calibrators.
Add 5 microliters of each solution to the corresponding labeled tube
containing 150 microliters of spotting solution.
4. Add 1 to 5 microliters of each test sample DNA to the remaining
tubes containing 150 microliters of spotting solution.
5. While wearing clean gloves, cut a piece of Biodyne B membrane to
11.0 centimeters by 7.9 centimeters. Cut a small notch in the upper
right corner of the membrane to mark orientation. Place the
membrane in the Hybridization tray containing 50 milliliters of Prewetting solution. Incubate at room temperature for 1 to 30 minutes.
6. Using forceps, remove the membrane from the Pre-wetting solution.
Place the membrane on the gasket of the slot blot apparatus, then
place the top plate of the slot blot apparatus on top of the membrane.
Turn on the vacuum source. Turn off the sample vacuum and
turn on the clamp vacuum on the slot blot apparatus. Push
down on the top plate to ensure the formation of a tight seal. Pour off
the Pre-wetting solution and rinse the Hybridization tray thoroughly
with deionized water.
7. Use a new pipette tip for each sample. Pipette each sample
(approximately 155 microliters) into a different well of the slot blot
apparatus. Slowly dispense each sample directly into the center of
each well of the slot blot apparatus ensuring that the pipet tip is
approximately 5 millimeters above the membrane.
52
This step must be witnessed.
8. After all samples have been pipetted into the wells of the slot blot
apparatus, slowly turn on the sample vacuum. Leave the sample
vacuum on until all of the samples have been drawn through the
membrane (approximately 30 seconds). Inspect each slot that contains
a sample for a uniform blue band. Turn off the sample vacuum.
Turn off the clamp vacuum. Turn off the vacuum source.
Disassemble the slot blot apparatus and remove the membrane.
Do not let the membrane dry out.
Clean slot blot apparatus following each use as follows: Soak the
slot blot apparatus in a large volume of dilute SDS solution for
approximately 5 to 15 minutes. Using a disposable lab towel, clean
the gasket and the side of the top plate that contacts the membrane.
Then rinse the slot blot apparatus with an excess of water and allow
to dry at room temperature. Never use bleach.
III. DNA Hybridization For Quantiblot
Principle: The DNA Samples immobilized on the nylon membrane must
next be hybridized to the biotinylated Quantiblot D17Z1 probe.
The hybridized samples are then bound to the Enzyme
Conjugate:HRP-SA followed by a stringent wash to remove nonspecifically bound probe.
The membrane must not be allowed to dry out at any time during
this protocol.
The Hybridization solution and the Wash solution must be warmed to
between 37o and 50oC in either a water bath or an incubator. All solids must
be in solution before use.
1. Pre-hybridization: Transfer the membrane to 100 milliliters of
pre-warmed Hybridization solution in the Hybridization tray. Add 5
milliliters of 30% hydrogen peroxide. Place the lid on the tray. Use a
weight to keep tray from floating in the water bath. Rotate in a 50oC
(+/- 1oC) water bath (50 to 60 rpm) for 15 minutes (+/- 2 minutes).
Pour off the solution.
53
2. Hybridization: Add 30 milliliters of Hybridization solution to the
Hybridization tray containing the membrane. Tilt the tray to one side
and add 20 microliters of Quantiblot D17Z1 probe to the Hybridization
solution. Place the lid on the tray. Rotate in a 50oC (+/- 1oC) water
bath (50 to 60 rpm) for 20 minutes (+/- 2 minutes).
Pour off the solution.
3. Rinse the membrane briefly in 100 milliliters of pre-warmed Wash
solution by rocking the tray for several seconds. Pour off the solution.
4. Stringent Wash/Conjugation: Add 30 milliliters of the prewarmed Wash solution to the Hybridization tray. Tilt the tray to one
side and add 90 microliters of the Enzyme Conjugate:HRP-SA to the 30
milliliters of Wash Solution. Place the lid on the tray. Rotate in a
50oC (+/- 1oC) water bath (50 to 60 rpm) for 10 minutes (+/- 1 minute).
Pour off the solution.
5. Rinse the membrane thoroughly for 1 minute in 100 milliliters of
pre-warmed Wash solution by rocking the tray or rotating it on a
orbital shaker (100 to 125 rpm) at room temperature. Pour off the
solution. Rinse again for 1 minute. Pour off the solution.
6. Wash the membrane by adding 100 milliliters of pre-warmed Wash
solution to the tray. Place the lid on the tray. Rotate at room
temperature on an orbital shaker (100 to 125 rpm) for 15 minutes.
Pour off the solution.
7. Rinse the membrane briefly in 100 milliliters of Citrate Buffer by
rocking the tray. Pour off the solution.
54
IV. Chemiluminescent Detection Steps For Quantiblot
Note: Chemiluminescent Detection Reagents should be stored
separately (at 2o to 8o) and not allowed to cross-contaminate each
other.
1. Mix 5 milliliters of each solution together. Do not prepare this
mixture more than 5 minutes before use. Add the 10 milliliters of
Chemiluminescent Detection reagent mixture to the membrane in the
Hybridization Tray and shake for 5 minutes at room temperature.
Pour off the solution.
Note: For maximum sensitivity, expose the membrane to X-ray film
within 10 minutes of incubation in the Chemiluminescent
Detection reagents.
2. Cut a piece of plastic-backed paper to approximately 9 x 12
centimeters. Place the damp membrane DNA side up on the plastic
coated side of the plastic-backed paper. Cover the membrane with a
piece of plastic wrap. Use a paper towel to smooth out any wrinkles or
air bubbles in the sides. Fold the plastic wrap around the rest of the
membrane.
3. In a darkroom, place a piece of X-ray film in the film cassette.
Carefully place the covered membrane on top of the film such that the
DNA side in contact with the film. Do not move the membrane
once it is placed on top of the film; movement may cause
blurring of the resulting image or a “double image”. Close the
film cassette. It is very important that the film is in tight,
uniform contact with the covered membrane.
4. Expose the film for approximately 5 minutes at room temperature.
5. Process the film with an automatic film processor.
V. Results Interpretation
Results are interpreted by comparing the signal intensity of the DNA
test sample to the signal intensity obtained for the DNA standards. The
signal intensity for a sample reflects the total amount of DNA spotted on the
membrane.
55
The DNA Calibrators are used to provide DNA of a known
concentration to verify that the DNA Standards were correctly diluted and
are providing correct results for the test samples. For example, the DNA
Calibrator 1 has a stock concentration of 0.7 nanograms/microliter. Five
microliters of this control was added to 150 microliters of Spotting solution
and the entire 155 microliters was spotted on the membrane. Thus, 3.5
nanograms of this sample was spotted on the membrane (0.7 ng/ul X 5 ul =
3.5 ng). The signal obtained for this control sample should have an intensity
that is between the 2.5 and 5 ng DNA Standards. Likewise, the DNA
Calibrator 2 should have an intensity that is between the 0.3125 and 0.625
ng DNA Standards.
The seven DNA Standards represent the following quantities of DNA
spotted on the membrane:
A = 10 ng; B = 5 ng; C = 2.5 ng; D = 1.25 ng; E = 0.625 ng; F = 0.3125
ng and G = 0.15625 ng. DNA Calibrator 1 should have an intensity that is
between DNA Standards B and C. DNA Calibrator 2 should have an
intensity that is between DNA Standards E and F. Quantities for the test
samples are determined by comparison of signal intensities to the DNA
Standards.
The concentration of a DNA test sample is determined as follows:
1. Determining the quantity of DNA test sample spotted on the
membrane by comparing its signal intensity to the intensity of the
DNA Standards.
2. Divide this quantity by the volume of DNA test sample added to the
spotting solution (typically 5 microliters of DNA test sample is added
to 150 microliters of spotting solution). This calculation gives DNA
concentration in ng/ul.
Example: The questioned sample compares favorably with the DNA
standard containing 10 ng of DNA. This sample would be equivalent to
10 ng/5 ul, since we added 5 microliters of input DNA for the testing process.
This is equal to 2 ng/1 ul. We want 1.0 ng of input DNA for the amplification
process. We will need to determine how many microliters of DNA we need to
add in order to provide a final concentration of 1.0 ng.
Therefore, we set up a ratio: 2 ng/1.0 ul = 1.0 ng/x ul. We cross multiply and
get: 2 ng (x ul) = 1.0 ng (1.0 ul). We then solve for x.
x ul = 1.0 ng (1.0 ul)
2 ng
56
x ul = ½ ul therefore, x = 0.5 ul of input DNA to provide a final concentration
of 1.0 ng of DNA for amplification.
Critical Aspects
and Limitations: Analysts should use their own discretion when
interpreting the results from the Quantitation procedure.
Very small samples with no result on the quantiblot
should be considered for amplification with extreme
caution.
57
DNA Amplification
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
This procedure has been taken from the AmpFiSTR Profiler Plus
Amplification Users Manual.
1. Determine the number of samples to be amplified, including controls.
Place the required number of tubes in a rack and label them
appropriately.
2. Vortex the reaction mix, the primer set and the DNA polymerase and
quick spin in a microcentrifuge to remove any liquid from the caps.
3. Prepare the master mix by adding the following volumes of reagents to a
microcentrifuge tube:
Number of samples x 21.0 ul of reaction mix
Number of samples x 1.0 ul of DNA polymerase
Number of samples x 11.0 ul of primer set
4. Mix this master mix thoroughly by pipetting up and down several times.
5. Dispense 30 ul of this master mix into each sample amplification tube.
6. Add 20 ul of sample to each tube.
DNA Test Sample tubes: Add 20 microliters of sample DNA.
Positive Control tube: Add 20 microliters of Control DNA 1.
Negative Control tube: Add 20 microliters of TE buffer.
Note: Each PCR amplification is performed in a final volume
of 50 microliters.
7. For each of the following additions, complete the processing of each tube
before proceeding to the next tube. No more than one tube should be
58
open at a time. Use a new pipet tip for each addition. Discard the pipet
tip and re-cap the tube before proceeding to the next sample.
8. Transfer tubes to a “carrier rack” and carry to the Thermal Cycler. Do
not set the carrier rack down.
9. Place the PCR reaction mix tubes into the Thermal Cycler. Push the
tubes down completely into the heat block.
Turn on the Thermal Cycler 2400.
Press Run
Select Profiler program and press Start
Choose Reaction volume of 50 microliters and press Start
Cycling parameters are:
Initial incubation step HOLD at 95oC for 11 minutes.
Denature at 94oC for 1 minute.
Anneal at 59oC for 1 minute.
Extend at 72oC for 1 minute.
Program for 28 cycles.
Final extension step HOLD at 65oC for 45 minutes.
Final step HOLD at 25oC for infinity.
10. After the amplification process, remove the samples from the Thermal
Cycler. Samples are now ready for DNA typing using the 310 Genetic
Analyzer or they may be stored at 4oC for 7 days, or at -20oC for extended
periods.
59
Preparation of Samples for 310 Genetic Analysis
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Materials:
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
310 Genetic Analyzer 0.5 ml sample tubes
310 Genetic Analyzer Septa for 0.5 ml sample tubes
Deionized Formamide
Genescan - 500 ROX Internal Lane Size Standard
0.5 milliliter microcentrifuge tubes
Plastic-backed paper
To prepare the samples for analysis:
1. Add the following reagents to a 1.5 microliter centrifuge tube in the
calculated volumes below.
1. GeneScan-500 (ROX) size standard
2. Deionized formamide
1 ul x (number of samples)
24 ul x (number of samples)
Mix solution by pipetting up and down several times.
Solution should be made fresh before each use.
2. Label an appropriate number of 0.5 milliliter samples tubes, including an
allelic ladder, positive control, and a blank control.
3. Aliquot 25 microliters of formamide/ROX solution into each of the sample
tubes.
4. Add 1.5 microliters of PCR product or allelic ladder to each tube and mix
the samples by pipetting up and down.
5. Seal each tube with a rubber septum.
6. Denature each sample by placing in a heat block for 3 - 5 minutes at 95oC.
7. Snap cool denatured samples for 3 - 5 minutes in an ice bath.
60
8. Samples are now ready for capillary electrophoresis.
61
Setting the Run Temperature
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
1. Close the instrument doors.
2. Launch the ABI Prism 310 Collection Software.
3. Under the “Window” pull down menu, select “Manual Control”
4. Under the “Function” pull down menu, select “Temperature Set”, enter a
value of 60 and choose “Execute”
It takes between 20 and 30 minutes for the instrument to reach 60oC. The
Sample Sheet and Injection list can be prepared during this warming up time
period.
62
Creating a Genescan Sample Sheet
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
1. Launch the ABI Prism 310 Collection Software.
2. Under the “File” pull down menu, select “New” to create a new file.
3. Select “Genescan Sample Sheet 48 Tubes” from the resulting window.
4. A blank template opens so that the samples can be entered into the
sample sheet.
5. Click on the sample box adjacent to A1 and type ladder. This sample will
represent the allelic ladder for the run.
6. Enter sample names/numbers for each injection in the sample name
column, beginning with A3. This will indicate which sample is in which
tube of the sample tray.
7. In the “Pres” (present) column, select all 4 dye colors.
8. A “Diamond” is the STD (standard) column indicates the size standard
used. We use ROX - R.
9. The “Sample Info” boxes must be filled in so that Genotyper will operate
properly.
A.
B.
C.
D.
E.
F.
Highlight all samples in the Sample Name column.
Select the “Edit” pull down menu.
Choose “Copy”
Highlight the “Sample Info” boxes.
Select the “Edit” pull down menu.
Choose “Paste”
63
10. Under the “File” pull down menu, select “Save As”.
11. Type the name of the sample sheet file and select “Save”.
64
Creating a Genescan Injection List
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
1. After creating a sample sheet file and saving it, open the “File” pull down
menu and select “New”.
2. Select “Genescan Injection List” from the resulting window.
3. To import the sample sheet information onto the injection list, choose the
appropriate sample sheet from the Sample Sheet pull down menu by
dragging the cursor through the pull down menu to the appropriate
“Saved” sample sheet.
4. Verify that the correct Module appears for each sample. (GS STR POP4 (1
ml) F).
5. The injection time should be set at 5 seconds per sample.
6. The injection voltage should be set at 15.0 kV.
7. The run voltage should be set at 15.0 kV.
8. The run temperature should be set at 60oC.
9. The run time should be set to 24 minutes.
10. Choose the appropriate Matrix from the pull down menu under the “Set
Matrix File” column. To do this, Click on the arrow in the Matrix column
to view the pop up menu of available matrices.
11. Deselect the boxes in the “Autoanalyze” column for each sample.
12. Verify that the analysis parameters indicate “Profiler/Cofiler”.
13. Verify that the sizing standard column lists “none”.
65
14. Deselect the auto print box.
15. Load the sample tubes into the sample tray in the correct order according
to the injection list.
16. The injection list does not need to be saved.
17. Select “Run” to start the 310.
66
Developing a Matrix File
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle: A matrix file is developed for the 310 capillary electrophoresis
unit so that the signals from each of the four dyes can be
isolated from each other during multicomponent analysis.
Materials:
310 Genetic Analyzer 0.5 ml sample tubes
310 Genetic Analyzer Septa for 0.5 ml sample tubes
Deionized Formamide
Perkin Elmer Dye Primer Matrix Standards
1. Close the instrument doors.
2. Launch the ABI Prism 310 Collection Software.
3. Under the “Window” pull down menu, select “Manual Control”
4. Under the “Function” pull down menu, select “Temperature Set”, enter a
value of 60 and choose “Execute”
5. Prepare the Matrix standards as follows:
A. Label 4 310 Genetic Analyzer 0.5 ml sample tubes with:
1. FAM
2. JOE
3. NED
4. ROX
B. Vortex and quick spin the stock tubes of dye.
C. Add 24 microliters of deionized formamide and 1 microliter of each
matrix standard to each of the appropriately labeled tubes.
D. DO NOT INCLUDE THE GENESCAN 500 ROX SIZE STANDARD
IN THE MATRIX SAMPLES.
E. Seal each tube with a rubber septum.
F. Denature each sample by placing in a heat block for 3 - 5 minutes
at 95oC.
G. Snap cool denatured samples for 3 - 5 minutes in an ice bath.
H. Samples are now ready for capillary electrophoresis.
6. Create and save a GeneScan sample sheet.
67
A. Under the “File” pull down menu, select “New” to create a
new file.
B. Select “Genescan Sample Sheet 48 Tubes” from the resulting
window.
C. A blank template opens so that the samples can be entered
into the sample sheet.
D. Enter the appropriate sample names (FAM, JOE, NED, and
ROX) in positions for each sample.
E. Deselect the diamonds in the STD Column
F. In the “Pres” (present) column, select all 4 dye colors.
G. The “Sample Info” boxes must be filled in so that Genotyper
will operate properly.
Highlight all samples in the Sample Name column.
Select the “Edit” pull down menu.
Choose “Copy”
Highlight the “Sample Info” boxes.
Select the “Edit” pull down menu.
Choose “Paste”
H. Under the “File” pull down menu, select “Save As”.
I. Type the name of the sample sheet file and select “Save”.
7. Create and save a GeneScan Injection List
A. After creating a sample sheet file and saving it, open the
File” pull down menu and select “New”.
B. Select “Genescan Injection List” from the resulting window.
C. To import the sample sheet information onto the injection
list, choose the appropriate sample sheet from the Sample
Sheet pull down menu by dragging the cursor through the
pull down menu to the appropriate “Saved” sample sheet.
D. Verify that the correct Module appears for each sample. (GS
STR POP4 (1 ml) F).
E. The injection time should be set at 5 seconds per sample.
F. The injection voltage should be set at 15.0 kV.
G. The run voltage should be set at 15.0 kV.
H. The run temperature should be set at 60oC.
I. The run time should be set to 24 minutes
J. Under the matrix file heading, each injection should be
“none”
K. Select “none” for the analysis parameters
L. Select “none” for the size standard
M. Deselect auto print
8. To begin the 310 Collection software, select “Run”
When injections are complete:
68
A. To name and save your completed run, choose “Save Project As”
under the “File” pull down menu.
B. Launch the GeneScan software
C. Under the “File” pull down menu, select “New”.
D. Choose the “Matrix” icon.
E. The “make new matrix” box appears.
1. Indicate the sample files that correspond to each matrix
standard dye color. For example, select the “B” icon and a
the sample file that corresponds is FAM.
2. Repeat this procedure for the remaining three dye samples.
3. Select a starting scan number of 3300 for each sample. This
starting scan number is intended to exclude the primer
peaks.
4. Enter a value of 2500 points.
5. Select “ok” and the matrix file table appears.
69
F. The number values appearing in the matrix table may vary from
matrix to matrix. In the boxes where the row matches the column,
(B and B), the numerical value should read 1.0000.
G. Under the “File” pull down menu, select “Save As”. Name the file
and select “ok” to save it.
Verify the accuracy of the Matrix File.
1. Open the matrix project file.
2. The “Analysis Control” window will open.
a. Select the all the sample files to be analyzed.
b. To install the new matrix, under the “sample” pull down
menu, select “Install New Matrix”. Choose the new matrix
file to be applied and select “open”.
c. No size standard should be applied for analysis of the matrix
samples.
d. Select the “Analyze” icon to analyze the matrix samples.
e. In the “Results Control” window, examine the results for all
four colors for each of the matrix standard samples.
Interpretation
The FAM matrix standard results should have peaks for Blue. The other
colors should have a relatively flat baseline. A pattern of pronounced peaks
or dips in any of the other three colors indicates that the color separation is
not optimal.
Examine the results for each matrix standard sample in this way.
70
Genescan Data Analysis
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
A. Setting the Analysis Parameters:
1. Launch the Genescan Analysis software.
2. Under the “File” pull down menu, select Analysis Parameters.
3. Fill in the Dialogue box with the settings below:
Analysis Range Box
Select “This Range”
Enter “Start” value of 3200
Enter “End” value of 6300
Data Processing Box
Select “Baseline” and “Multicomponent”
“Smooth Options” select “Light”
Peak Detection Box
Select 75 as the Peak Amplitude Threshold for each color
Size Call Range Box
Select “All Sizes”
Size Calling Method Box
Select “Local Southern Method”
Split Peak Correction Box
Select “none”
4. Under the “File” pull down menu, select “Save As” and save these
parameters as Profiler/Cofiler
5. Choose “OK”
71
1. Under the “Settings” pull down menu, choose “Auto-analysis
defaults”. Once the auto-analysis defaults are set and saved they
will not need to be adjusted.
Deselect “Always Override Collection Settings”
for “Standard” select “none”
for “Dye” select “R (red)”
for “Parameters” select “Profiler/Cofiler”
Deselect “Auto-print” box
Choose “ok”
7. Under the “Settings” pull down menu select “Preferences” and
“Results Display”. Once the preferences for results display are set
and saved they should not need to be adjusted.
Default Display Attributes Box:
Select “Align by Size,”
Select “Show Legends”
Select Show Offscale Regions
Select “Standard” Plot Colors
Select “Transparent Peak Highlighting”
Printing Preferences Box:
Select “As Shown on Screen”
Stacked Electropherogram Panels
deselect “Use Common Vertical Scale”
enter “2.5” for the minimum panel height
72
enter “14.0” for the maximum panel height
B. Creating a size standard: A size standard should be created with
each new run.
1. In order to create a size standard, select all samples in the project
folder that contain an internal size standard.
2. Under the “file” pull down menu choose “new”
3. Choose the “Size Standard” icon.
4. A window will open prompting you to open a file from your results
folder. Apply one size standard to all injections, therefore open an
allelic ladder sample to define as a size standard.
5. Another window opens prompting you to define the “Dye and
Analysis Parameters”
Select “R (red)”
Select “Profiler/Cofiler”
Choose “ok”
73
6. A window will open so that the peaks of the size standard can be
defined.
To enter values, place the cursor inside the peak and select
the peak. The peak will become highlighted and a box will
open in the chart down below for a value to be entered.
After the value is entered, press return.
7. Locate the triplet of peaks and label them “139”, “150”, and “160”.
8. The next value is “200”.
9. Do not label the next peak.
10. The final peaks are to be labeled “300”, “340”, “350”, “400”, “450”,
“490”, and “500”.
11. The first size peak is often lost within the primer dimer front
therefore the remaining peaks should be labeled from where the
baseline is stable. (35, 50, 75, and 100)
74
12. Under the “file” pull down menu choose “save as”
13. Save the file as “Size Standard : Date” and close the size standard
window.
14. Relaunch the genescan file that you were working on and, define
the size standard in the column labeled “Size Standard” in the
“Analysis Control Window” and apply the new size standard to the
samples in the project file.
15. Define the analysis parameters in the column labeled “Parameters”
in the “Analysis Control Window” and apply “Profiler/Cofiler” to the
samples in the project file.
16. Select all the samples to be analyzed by clicking on the top left
space (above the number column). This selects all four colors for all
of the sample files.
17. Choose the “Analyze” button. This fills the sample files with the
analyzed data from each injection.
18. After the analysis is complete, confirm that the sizes for the peaks
have been correctly assigned.
A. Under the “window” pull down menu, choose “Results
Control”
B. Select “1” electropherogram panel for display
C. Select “R” (red) for all the injections to examine the red
peaks in overlapping groups
D. Select “on” in the Quick Tile Box
1. All the peaks should line up with each other
2. Peak intensity should be between 1500 to 4500
relative fluorescent units.
3. Scroll through the tables to verify the peak
assignments for the data.
4. Close this window and select “Clear All” from the
75
“Results Control” window.
19. To examine the data for each color of individual sample files:
A. Select “8” electropherograms panels for display
B. Select sample 1 to examine the data for each color in an
individual panel.
C. Select “on” in the Quick Tile Box
20. For data comparison of peak heights, the vertical scale should be
adjusted.
A. Place the pointer over the vertical number scale for a sample
and double click
B. A window will open prompting you to fill in the “tick spacing”
and the size range of data for display.
C. Select the box to apply this scale to all electropherogram
panels and choose “ok”.
D. Check the peaks in the remaining samples.
C. Results and Interpretation
After the sample files have been analyzed, the results control window is used
to display the results from each injection into a capillary. This window
displays the newly analyzed sample files and allows the user to specify a
format of results. Selecting both the electropherogram and the tabular data
is recommended for reviewing the results.
The electropherogram is a chromatographic display with fluorescence
intensity indicated as relative fluorescence units on the y-axis. After the
internal size standard has been defined and applied, the electropherogram
can be displayed with the base pair size on the x-axis.
76
Peaks of all heights within the analysis range specified in the analysis
parameters are displayed on the electropherogram, but those peaks below the
peak amplitude threshold defined in the analysis parameters will not be
listed on the tabular data and therefore are not imported into the genotyping
program.
Critical Aspects
and Limitations: For samples with low peak heights, the injection time
may be changed from 5 seconds to up to and including 15
seconds based on the analyst’s discretion. For samples
with high peak heights, the injection time may be
decreased from 5 seconds to as short as one second.
Analysis time may also be changed to accommodate room
temperature. Run time may vary from 24 minutes to up
to 26 minutes based upon the temperature of the DNA
amplification room.
The RFU threshold for the 310 Genetic analyzer at the
Hamilton County Coroner’s Laboratory is set at 100
RFU’s. This is based on a value of three times the noise
level (determined previously to be approximately 20 - 30,
by the application specialist from Applied Biosystems).
This value for noise was recalculated following NT
validation of the 310 and determined to be approximately
35 RFU’s. If necessary, the RFU threshold may be
lowered due to decreasing sensitivity of the instrument
over time. This may be due, in part, to an aging laser or
camera.
A sample may be reinjected following a run without
rerunning the control samples.
The changes in analysis parameters will not need to be
validated by the Hamilton County Coroner’s Laboratory
since they do not represent a significant departure from
the established protocol.
77
Genotyper Data Analysis
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised: 2/23/04
Principle: The Genotyper software is used to convert allele sizes obtained
from the GeneScan software into allele designations. Genotypes
are assigned by comparing the sizes obtained for the unknown
sample alleles with the sizes obtained for the alleles in the
allelic ladder.
1. From the desktop, launch either the Profiler Plus or the Cofiler program
and the appropriate genotyper window will open.
2. Under the file pull down menu, choose “Import GeneScan File (s)” and a
window will open prompting you to import your data.
3. Verify that the “Run Folder” is displayed and open, and select the
GeneScan Project you are interested in importing into Genotyper.
4. Import the entire GeneScan project or import only select files of interest.
5. Under the “Macro” window, select “Kazam” and double click. This will
initiate the Genotyper Macro to begin analyzing the data.
78
Interpretation
The following information is intended to be used by the Forensic Scientist as
a set of guidelines. The interpretation of data in each case falls to the
discretion of the Forensic Scientist.
Genotypes are assigned to sample alleles by comparison of their sizes to those
obtained for the known alleles in the allelic ladders. Allele categories are
defined to be +/- 0.5 base pairs (bp) wide. Peaks that size within +/- 0.5 bp of
an allele category will have a label indicating the allele designation.
Peaks that do not size within an allele category will have a label indicating
“OL Allele?”
A sample allele peak must have been “recognized” in GeneScan before it can
be recognized by Genotyper. Thus, sample allele peaks that are below the
Peak Amplitude Threshold that was specified in the GeneScan analysis
Parameters cannot be labeled in Genotyper.
Assessment of Successful Amplification and Electrophoresis
The internal size standard GeneScan ROX-500 must have correct sizes
assigned to the peaks used for sizing in the analytical range of 75-400 bp.
The 250 bp peak is not used for sizing purposes.
The positive amplification control must type correctly at all loci.
Negative amplification controls and reagent blank controls must not exhibit
reproducible peaks greater than 150 RFU in any of the dyes within the size
ranges covered by any of the loci. Please refer to page 106 of this manual for
interpretation guidelines.
Examine each sample for any extraneous peaks which are present at a given
base pair size in two or more dyes. This may indicate problems with the
integrity of the polymer (spikes) for that sample. Labels from this peak may
be ignored but may be noted on the print out and reason given.
Examine the allelic ladder used for genotyping to determine that all allele
designations have been assigned correctly by the Genotyper program.
Examine each sample for any peaks present at a given base pair size in one
or more dyes which echo the presence of a relatively large peak at that same
base pair size in another one of the dyes. This may be an indication of
fluorescent pull-up and might result in the pull-up peak being given an allele
79
designation. Labels from this peak may be ignored and may be noted on the
print out and reason given.
The internal size standard should have a 400 base pair peak. However, the
amplification and typing room at the Hamilton County Coroner’s Lab has
large temperature fluctuations. This may cause the 400 peak to be absent.
Interpretation is at the discretion of the analyst.
Allele Definition:
For any given locus, minor peaks that are above 75 RFU and are located at a
position one repeat smaller than a major peak must be evaluated as to
whether it represents stutter or a real allele. Such peaks may be considered
as stutter if their peak height percentages to the larger peaks is equal to or
less than 10%. Stutter percentages reported in the Profiler Plus and Cofiler
procedure manuals may also be consulted. The Hamilton County Coroner’s
Lab uses 10% for any stutter calculations.
For any given locus, minor peaks that are above 75 RFU and are located at a
position one repeat unit smaller than a major peak may be considered as a
real allele if their peak heights exceed 10%.
For any given locus, minor peaks above 75 RFU with no major peaks at a
position one repeat unit larger to it will interpreted as a real allele unless the
results of the complete profile cause that particular allele to be judged
inconclusive.
True off ladder alleles or variants may be verified by re-injecting the sample
and demonstrating that the result is not an artifact. The Genotyper data can
also be checked to see if the allele in question falls near one of the designated
bins for that locus. Determination of the authenticity of the allele in question
can then be made by the Forensic Scientist.
Peaks for questioned stains and for reference samples should fall between 75
and 6000 RFU. However, due to a large amount of amplification product,
peaks larger than 6000 RFU can occur. Interpretation of peaks in excess of
6000 RFU is at the discretion of the Forensic Scientist.
80
Single known profile If the peak heights of two alleles are within a minimum
of 70% of one another and they are of at least 75 RFU, they can be considered
to be a heterozygote pair.
Sample quality and quantity for amplification may result in deviations from
the 70% model. Such results may be called inconclusive. Degraded samples
may produce results which fall outside the 70% model for interpretation.
These samples will be interpreted with caution at the analyst’s discretion.
Mixed or unknown profile If peak heights of two alleles are within 70% of
one another and they are of at least 75 RFU, they can be considered a
heterozygote pair.
If the peak height of the smaller allele is less then 70% of the major peak the
alleles may represent a mixture from separate sources. Such a mixture may
be confirmed by at least one 3 allele locus present in the profile.
Three alleles at any locus is indicative of a mixture.
Minor and major alleles may be assigned through the use of the heterozygote
model of 70%.
Example #1 of stutter and peak height ratio evaluation
81
Allele
RFU
28
582*
30
287
31
560*
32.2
259
*known allele
Minus Stutter(10%)
231
534
1. Compensate for stutter by subtracting 10% of adjacent larger alleles from
the smaller alleles.
560 - 26 = 534
287 - 56 = 231
2. Do the two known alleles fit into the 70% model?
534/582 x 100 = 92% Yes
3. Do the two unknown alleles fit into the 70% model?
259/231 x 100 = 90% Yes
Example #2 of stutter and peak height ratio evaluation
Allele
RFU
Minus Stutter(10%)
9
491
11
212*
150
12
626*
*known allele
1. Compensate for stutter by subtracting 10% of adjacent larger alleles from
the smaller alleles.
212 - 62 = 150
2. Do the two known alleles fit into the 70% model?
150/626 x 100 = 24% No
3. What could be the possible peak height for a second unknown? (What is
70% of the unknown peak?)
491 x 0.70 = 343.7 and 491/0.70 = 701. Range = 344 - 701.
4. Subtract out the possible sister allele of the unknown (9) from the first
known allele (11).
150 - 343 = Impossible
5. Subtract out the possible sister allele of the unknown (9) from the second
known allele (12).
626 - 343 = 283
626 - 491 = 135
626 - 701 = Impossible
82
6. Now, do the two known alleles now fit the 70% model?
150/283 x 100 = 53% No
135/150 x 100 = 90% Yes. Therefore, the unknown pair is 9, 12.
83
Chelex
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
Chelex solution is used in the extraction of DNA. The
presence of chelex during extraction prevents the
degradation of DNA by chelating the metal ions that may
act as catalysts in the breakdown of DNA.
Materials:
Chelex-100 Resin
Reagent
Preparation:
5% (w/v), 100 milliliters
Weigh out 500 milligrams of Chelex-100 resin into a
sterile 15 milliliter tube. Add 10 milliliters of autoclaved,
deionized water. Make fresh for each use.
84
Citrate Buffer
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
The citrate buffer is used as part of the color development
process for DNA quantitation and for DNA typing. It is
used to lower the pH of the system so that the colored
product will precipitate onto the nylon membranes for
quantitation and typing.
Materials:
Trisodium citrate, dihydrate
Citric acid, monohydrate
Reagent
Preparation:
Dissolve 18.4 grams of trisodium citrate, dihydrate in 800
milliliters of deionized water. Adjust the pH to 5.0 (+/0.2) by adding approximately 6 grams of citric acid
monohydrate. Adjust to a final volume of 1 liter using
deionized water and mix thoroughly. Store at room
temperature.
85
Dithiothreitol
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
1 M dithiothreitol solution is used in the extraction of
DNA. It lyses the sperm head and facilitates protein
denaturation.
Materials:
Dithiothreitol
Reagent
Preparation:
Dissolve 1.54 grams of dithiothreitol in 10 milliliters of
sterile deionized water in a sterile disposable plastic 15
milliliter tube. DO NOT AUTOCLAVE. Store 100
microliter aliquots in sterile 0.5 milliliter microfuge tubes
at -20oC. Discard any unused portion of a thawed tube.
86
0.5 M EDTA Solution
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
General:
Ethylenediamine Tetraacetic acid chelates magnesium
ions so that nucleases cannot chew apart DNA.
Materials:
EDTA
Sodium hydroxide
10N Sodium hydroxide
Reagent
Preparation:
Add 93.05 grams of EDTA to 400 milliliters of deionized
water. Stir vigorously on a magnetic stirrer. To dissolve
the EDTA powder, adjust the pH to 8.0 (+/- 0.2) by adding
approximately 10 grams of NaOH pellets. Check the pH
and add 10N NaOH if further pH adjustment is needed.
Bring volume up to 500 milliliters with deionized water.
Autoclave the solution and store at room temperature.
87
Genetic Analyzer Buffer 1X Concentration
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
General:
Genetic Analyzer Buffer with EDTA is used during the
capillary electrophoresis of samples in the 310 Genetic
Analyzer.
Materials:
Deionized water
10X Genetic Analyzer Buffer with EDTA
Reagent
Preparation:
Mix 1.5 milliliters of 10X Genetic Analyzer Buffer with
EDTA and 13.5 milliliters of deionized water.
88
Hybridization Solution for Quantiblot
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
The hybridization solution is the solution used to allow
the DNA probe D17Z1 to hybridize with the DNA sample
that has been extracted.
Materials:
20X SSPE
20% SDS
Reagent
Preparation:
Add 250 milliliters of 20X SSPE and 25 milliliters of 20%
w/v SDS to 725 milliliters of deionized water.
Hybridization solution solids must be in solution before
use. Warming the solution in a water bath will help the
solids to dissolve completely.
89
Phosphate Buffered Saline
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Materials:
Reagent
Preparation:
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Potassium Chloride
Sodium Chloride
Potassium Phosphate, monobasic
Anhydrous Disodium Phosphate
Dissolve 0.1 grams of potassium chloride, 4 grams of
sodium chloride, 0.1 grams potassium phosphate
monobasic, and 1.1 grams anhydrous disodium phosphate
in 400 milliliters of deionized water. Adjust pH of
solution to 7.4 if necessary. Adjust to a final volume of
500 milliliters using deionized water. Sterilize by
autoclaving and store at 2o to 8oC.
90
Prewetting Solution for Quantiblot
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
The prewetting solution is used to moisten the Biodyne B
membrane prior to applying the membrane to the slot blot
apparatus.
Materials:
5 N Sodium Hydroxide
0.5 M EDTA
Reagent
Preparation:
Add 40 milliliters of 5 N NaOH and 25 milliliters of 0.5 M
EDTA to 435 milliliters of deionized water and mix
thoroughly.
91
Proteinase K
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
CAUTION: POWDERED PROTEINASE K AND SOLUTIONS OF
PROTEINASE K CAN BE IRRITATING TO MUCOUS
MEMBRANES. WEAR SAFETY GLASSES AND GLOVES WHEN
HANDLING.
Principle:
Proteinase K is an enzyme that degrades Dnase and
removes proteins and contaminants from extracted DNA.
It chews up proteins in the membrane causing holes in
the membrane. Fluid (PBS, water) rushes in and bursts
the cells.
Materials:
Proteinase K
Reagent
Preparation:
Dissolve 100 milligrams of Proteinase K in 10 milliliters
of sterile deionized water in a sterile disposable plastic 15
milliliter tube. Store 100 microliter aliquots in sterile 0.5
milliliter microfuge tubes at -20oC. Thaw tubes as
needed. Discard any unused portions of thawed tubes.
92
20% Sarkosyl
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
Sarkosyl solution is used during extraction of DNA as a
protein denaturing detergent.
Materials:
N-Lauroylsarcosine
Reagent
Preparation:
Add 20 grams of N-Lauroylsarcosine to deionized water
and stir until dissolved. Bring to a final volume of 100
milliliters with deionized water and sterilize by passage
through a sterile 0.45 micrometer filter. Aliquot into
microcentrifuge tubes and store at room temperature.
93
20% (w/v) Sodium Dodecyl Sulfate
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
CAUTION: WEAR A MASK WHEN WORKING WITH
POWDERED SDS.
Principle:
Sodium dodecyl sulfate solution is a protein denaturant.
It acts to lyse the cell wall, denature enzymes and
dissociate nucleic acids from proteins. Within the DNA
complex it acts to solubilize the membranes of the nuclei.
Materials:
Sodium dodecyl sulfate
Reagent
Preparation:
Slowly dissolve 100 grams of sodium dodecyl sulfate in
400 milliliters of deionized water. To aid in dissolution,
solution may be warmed. Adjust to a final volume of 500
milliliters using deionized water and mix thoroughly.
Store at room temperature.
94
10N Sodium Hydroxide
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Materials:
Reagent
Preparation:
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Sodium hydroxide
Dissolve 400 grams of sodium hydroxide pellets in
approximately 700 milliliters of deionized water. Adjust
volume to 1.0 liter. Store at room temperature.
1.0 N Sodium hydroxide
Combine 100 milliliters of 10N sodium hydroxide with
900 milliliters of deionized water. Store at room
temperature.
95
Sperm Wash Buffer
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
The sperm wash buffer is used to wash away any extra
cellular material away from the sperm cells during the
extraction of semen stains.
Materials:
Sodium Chloride
1.0 M Tris-HCL
0.5 M EDTA
20% SDS
Reagent
Preparation:
Add 5 milliliters of 1 M Tris-HCL, pH 7.5, 10 milliliters of
0.5 M EDTA pH 8, 0.29 grams NaCl, 50 milliliters 20%
SDS to 430 milliliters of deionized water. Check pH.
Store at room temperature. Aliquot amount needed for
appropriate number of extractions into sterile disposable
50 milliliter plastic tubes and discard unused portion of
aliquot.
96
Spotting Solution for Quantiblot
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
This solution is mixed with DNA samples and is “spotted”
onto the slot blot membrane.
Materials:
5 N Sodium Hydroxide
0.5 M EDTA
Bromothymol Blue
Reagent
Preparation:
Mix together 750 microliters of 5N NaOH, 470 microliters
of 0.5 M EDTA, 18.75 microliters of 0.04% bromothymol
blue and 8.125 milliliters of deionized water. Store at
room temperature. Solution is stable for 3 months.
97
20X SSPE Buffer
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
This is a sodium chloride, sodium dihydrogen phosphate
and sodium EDTA buffer used in hybridization and blot
stripping.
Materials:
Sodium chloride
Sodium phosphate, monobasic
Disodium EDTA
10N Sodium hydroxide
Reagent
Preparation:
Dissolve 7.4 grams of disodium ethylenediamine
tetraacetic acid, dihydrate in 800 milliliters of deionized
water. Adjust the pH to 6 (+/- 0.2) with 10N sodium
hydroxide. Add 210 grams of sodium chloride and 27.6
grams of sodium phosphate, monobasic, monohydrate.
Adjust the pH to 7.4 (+/- 0.2) with 10N NaOH
(approximately 10 milliliters). Adjust to a final volume of
1 liter using deionized water and mix thoroughly.
Autoclave solution and store at room temperature.
98
Stain Extraction Buffer
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Materials:
Reagent
Preparation:
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Sodium Chloride
20% SDS
1.0 M Tris
0.5 M EDTA
Dissolve 1.46 grams of sodium chloride in 125 milliliters
of deionized water. To this solution add 2.5 milliliters of
1.0 M Tris, 5 milliliters of 0.5 M EDTA, and 25 milliliters
of 20% SDS. Titrate this solution to pH 8 with HCL.
Bring to a final volume of 250 milliliters with deionized
water. Store at room temperature.
99
TE Buffer
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
This buffer is used to resolubilize DNA at 56oC.
The EDTA is included to bind magnesium ions(Mg++).
Materials:
1.0 M Tris-HCL
0.5 M EDTA
Reagent
Preparation:
Add 5 milliliters of 1.0 M Tris-HCL, pH 8.0 (+/- 0.2) and
100 microliters of 0.5 M EDTA to 495 milliliters of
deionized water and mix thoroughly. Dispense 1
milliliter aliquots into microcentrifuge tubes and store at
room temperature.
100
Tris/EDTA/NaCl (TNE) Buffer
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
TNE buffer is used to extract the DNA from vaginal stain
mixtures.
Materials:
1.0 M Tris-HCL
0.5 M EDTA
1.0 N Sodium Hydroxide
Sodium Chloride
Reagent
Preparation:
Add 1 milliliter 1 M Tris-HCL to 75 milliliters of
deionized water. To this solution, add 0.584 grams of
sodium chloride and 200 microliters of 0.5 M EDTA. Stir
until dissolved. Adjust the pH to 8 with 1.0N sodium
hydroxide and bring to a final volume of 100 milliliters
with deionized water. Autoclave and store at room
temperature.
101
1.0 Tris-HCL
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Materials:
Reagent
Preparation:
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Trizma base
Concentrated HCL
Dissolve 30.27 grams of Tris base in 200 milliliters of
deionized water. Adjust to pH 8.0 (+/- 0.2) at room
temperature by adding approximately 11.25 milliliters of
concentrated HCL. Adjust the final volume to 250
milliliters and mix thoroughly. Autoclave and store at
room temperature.
102
Wash Solution for Quantiblot
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Principle:
The wash solution is used in the Quantiblot procedure for
washing the Biodyne B membrane following the DNA
hybridization step.
Materials:
20X SSPE
20% SDS
Reagent
Preparation:
Add 150 milliliters of 20X SSPE and 50 milliliters of 20%
w/v SDS to 1,800 milliliters of deionized water. Wash
solution solids must be in solution before use. Warming
the solution in a water bath will help the solids to dissolve
completely.
103
8. Case Work Documentation, Interpretation, Report
Writing and Review
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised: 2/23/2004
Laboratories should have policies, checks and balances in place which ensure
the reliability and completeness of the documentation, data analysis, reports
and review process.
8.1 Case Work Documentation
Documentation must be in such a form that a competent DNA analyst,
in the absence of the primary DNA analyst, would be able to evaluate
what was done and to interpret the data. Case documentation
includes all data obtained through the analytical process as well as
any pertinent notes regarding evidence packaging and condition. The
procedure ensures the preservation of documentation of procedures,
standards and controls used, observations made, results of tests
performed, photographs, electropherograms, communications, etc.,
which are used to support the DNA analyst’s conclusions.
8.2 Reading and Interpretation of the STR Data
All STR typing data will be “double read” to increase objectivity in the
interpretation of borderline results. The double reading will be done by a
“qualified second reader” (typically the other serologist/DNA analyst) in the
section.
Double reading will be done at the time of the technical review process.
Second readers are to check the data interpretations of the first reader, and
will initial the allele call sheet to document their findings. Any significant
differences in results obtained by the two readers must be resolved prior to
issuing a final interpretation and report. The two analysts will discuss their
findings and determine if there is a basis for agreement. The fact of and
rationale for any resulting change in interpretation is to be documented in
the notes. If, after discussion and review of the data, disagreement remains
regarding a particular result, the result is to be reported as inconclusive.
Interpretation of the results within the context of the case is the
responsibility of the primary analyst. The primary analyst is also
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responsible for verifying that consistent results were obtained by the second
reader and/or for identifying and addressing any differences.
8.2.1 Evaluation of Controls
8.2.1.1 Guidelines for interpreting and acting upon positive
and/or negative control results.
A. Reagent Blank
The reagent blank is a check for the possible contamination of
the sample preparation reagents by other human DNA or by
amplified DNA product. The reagent blank is performed by
carrying out the DNA extraction on a tube containing no
sample. This “blank” extract is then amplified and typed along
with the test samples. If there are any peaks present in this
sample that are greater than 150 relative fluorescence units
that cannot be attributed to being a spike, a pull up peak, or
noise, and the peaks follow the pattern of a DNA profile, the test
will be considered inconclusive. The samples accompanying this
reagent blank will be reextracted and the test repeated.
B. Amplification Blank (negative control)
The amplification blank is an additional check for
contamination during set up of the PCR reaction. It essentially
monitors the “environment” in that process for possible sources
of contamination. If there are any peaks present in this sample
that are greater than 150 relative fluorescence units that cannot
be attributed to being a spike, a pull up peak, or noise, and the
peaks follow the pattern of a DNA profile, the test will be
considered inconclusive. The samples accompanying this
amplification blank will be reamplified and the test repeated.
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C. Positive Control
The Profiler Plus and Cofiler control DNA provided in the
typing kits are positive controls which are used with each set of
samples typed to demonstrate that the kits are performing
properly. Each test will be considered inconclusive if these
controls do not show the proper type, or are blank.
8.2.1.2 Guidelines for statistical monitoring of the human DNA
control is appropriate. This is used only for RFLP analysis.
8.2.2 Evaluation of Samples
8.2.2.1 A sample will be called inconclusive if the “+” control,
the “-” control or the reagent blank controls do not perform as
expected.
Mixed samples will be interpreted with care. The major and
minor components will be reported as long as the minor
component alleles are greater than 75 relative fluorescent units
and cannot be attributed to spikes, pull up peaks, or stutter.
The 70% Rule discussed on page 82 of this manual will be
employed to determine heterozygote pairs.
A questioned sample will be called a match with a known
sample if the alleles present in the questioned sample are also
present in the known sample, with no extra alleles. A
questioned sample will be called a non-match with a known
sample if alleles are present in the questioned sample which
differ from the alleles present in the known sample. Likewise, if
alleles are present in the known sample which differ from the
alleles present in the questioned sample, the sample will be
reported as a non-match.
A sample with apparent degradation, that produces STR results
at less than 4 loci should be reported as insufficient for DNA
STR typing results.
8.2.2.2 This section deals with RFLP.
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8.2.2.3 Statistical Evaluation
The frequency of occurrence for the DNA profile will be
calculated using both the multiplication (or product) rule and
the NRC II report, which utilizes the value theta  for
homozygotes.
The Genotype frequencies for all loci are calculated as follows:
a. Heterozygote: use 2pq; Homozygote: use p2 + p(1-p), where
 = 0.01.
b. Genotype frequencies obtained for a sample at all thirteen
loci are multiplied to obtain a combined frequency estimate.
The Hamilton County Coroner’s Lab uses the Popstats program
which, was administered by the FBI. The Popstats program
uses population databases compiled by the FBI.
When reproducible off-ladder variant alleles are observed at a
locus, the locus will not be included in the frequency calculation.
When triallelism is observed at a locus, the locus will not be
included in the frequency calculation.
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8.3 Report Writing
Typical DNA reports should be structured as designed by the Hamilton
County Coroner’s Office. However, it is recognized that some unusual cases
may require special report structure. The following should be contained in
all final reports:
8.3.1 Case Identifier
The Crime Laboratory number must be included on all reports.
8.3.2 Identity of Analyst
The Analyst who worked the case must have their name and
signature an all reports.
8.3.3 Date of report
8.3.4 The DNA locus
The DNA locus must be specified for each detected sample. This
may be reported as “the thirteen loci approved by the FBI
CODIS program”.
8.3.5 Description of methodology
This may be indicated by reporting that STR DNA typing was
performed.
8.3.6 Results
Results, or lack thereof, must be included for all samples
analyzed.
8.3.7 Conclusions
Conclusions, or lack thereof, must be included for all samples
analyzed.
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8.3.8 Statistical Evaluation
Frequency of occurrence data should be included for all
probative samples with a reported type.
8.3.9 Signature of the reporting analyst
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8.4 Review
To ensure the proper adherence to protocol and QA standards in all aspects of
the typing and reporting procedure, data, documentation and reports are
reviewed independently according to the following procedure. Prior to
issuing a report, the reviewer(s) and the DNA analyst must agree on the
interpretation of the data and the conclusions derived from that data.
8.4.1 Analysis is Completed by DNA Analyst
A. Population frequencies are generated for each questioned
sample.
B. The case is written up according to Hamilton County
Coroner’s Office Guidelines.
8.4.2 Case is given to Evidence Technician staff for typing.
8.4.3 Case is given to Laboratory Director for review.
A. Approval of the case/results is indicated by initialing on the
handwritten copy of the report. In the absence of the Laboratory
Director, the analyst will perform an Administrative Review
according to instruction in section 2.6 of the Administration
Manual.
B. Case report is proofread for typographical errors and
returned to the secretary to print out on letterhead paper.
8.4.5 Case returned to DNA Analyst
Corrections are made, if necessary.
8.4.6 Case is Technical Reviewed by the second DNA Analyst
A. For all DNA cases, paperwork and notes are reviewed with
special attention given to conclusions and population
frequencies. The Technical Review form is completed and placed
in the case file upon completion of the technical review.
B. Approval of the case/results is indicated by completion of the
Technical Review form and by initialing on the DNA Typing
allele call sheet.
8.4.7 Report is returned to Evidence Technician staff for distribution.
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9.0 Proficiency Testing
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised: January 16, 2003
Proficiency testing is used regularly and periodically to demonstrate the
quality performance of the DNA laboratory and serves as a mechanism for
critical self-evaluation. This is accomplished by the analysis and reporting of
results from appropriate biological specimens submitted to the laboratory as
open and/or blind case evidence.
All specimens submitted as part of an open or blind proficiency test must be
analyzed and interpreted according to the DNA analysis protocol approved by
the laboratory for use at the time of the proficiency test.
Since the proficiency testing program is a critical element of a successful QA
program, it is an essential requirement. The Hamilton County Coroner’s
Laboratory Serology section has established its own proficiency testing
program, through the use of random samples, which verifies performance on
a yearly basis.
The Hamilton County Coroner’s Laboratory Serology section also participates
in proficiency testing programs, conducted by outside institutions, which are
appropriately and specifically designed for forensic DNA analysis. The
laboratory currently subscribes to Collaborative Testing Service, Inc.
9.1 Open Proficiency Testing
Open proficiency test specimens are presented to the laboratory and its staff
as proficiency specimens and are used to demonstrate the reliability of the
laboratory’s analytical methods, as well as the interpretive capability of the
DNA Analyst. Participation in the open proficiency test program is the
primary means by which the quality performance of this DNA laboratory is
judged and is an essential requirement since this laboratory performs case
work.
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9.1.1 Personnel
Open proficiency testing pertains to those DNA Analysts
actively engaged in DNA testing.
It is mandatory that the DNA Analyst conduct the entire test
alone, without selecting or accepting any assistance from other
persons. (This does not include processes that are performed in
batch, such as amplification and Quantiblot.) If DNA Analysts
have any questions or require assistance, they should contact
the Laboratory Director.
9.1.2 Frequency
Open proficiency tests are submitted such that each DNA
Analyst is tested at least twice a year.
9.1.3 Specimens
Each open proficiency test may consist of dried specimens of
blood and/or other physiological fluids, either singly or as a
mixture. Each sample to be tested should contain an amount
sufficient so that a conclusion can be drawn from the results of
the analysis.
9.1.4 Sample Preparation, Storage and Distribution
A. All specimens and proficiency tests should be uniformly
prepared using materials and methods that ensure their
integrity and identity.
B. All open proficiency test specimens are prepared on washed
cotton cloth, cotton swabs or other suitable material.
C. Each specimen and set is to be labeled with a unique
identifier.
D. A portion of each specimen used to prepare the open
proficiency test should be retained by the preparing laboratory
for possible referee analysis and comparison if circumstances
dictate.
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E. The Laboratory Director is responsible for acknowledgment
of receipt of each proficiency test and will assign it to the DNA
laboratory staff.
9.3 Documentation of Proficiency Test Results
9.3.1 Open Proficiency
Upon completion of a proficiency test, at a minimum, the following
proficiency test data and information should be collected and retained
and the results submitted to the Laboratory Director for evaluation:
1. Proficiency Test Set Identifier
2. Identity of DNA Analyst
3. Dates of Analysis and Completion
4. Copies of all Work Sheets and Notes
5. Electropherograms
6. Statistics for samples (when necessary)
7. Results/Conclusions
9.4 Review and Reporting of Proficiency Test Results
The review for all test materials is the same as that for a case. The results
will be reported, sent to the clerical staff for typing, sent for review by the
Laboratory Director, sent to the analyst for proofreading and then sent to the
submitting proficiency testing agency. All original notes, records, and other
data pertaining to the open proficiency test results are retained in a separate
filing system within the Serology section.
9.5 Corrective Action
The following clearly defines the specific policies, procedures and criteria for
any corrective action taken as a result of a discrepancy in a proficiency test.
9.5.1 Authority and Accountability
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It is the responsibility of the Laboratory Director to assure that
discrepancies are acknowledged and that any corrective action is
documented.
9.5.2 Administrative Error
Any significant discrepancy in a proficiency test determined to be the
result of administrative error (clerical, sample confusion, improper
storage, documentation, etc.) will be corrected through normal
laboratory practices. The Laboratory Director will initiate an
investigation to determine the root cause of the administrative error.
That investigation may result in modifications to laboratory
procedures, additional training, or changes to laboratory practices.
The Laboratory Director will be responsible for documenting corrective
actions and assessing their effectiveness.
9.5.3 Systemic Error
Any significant discrepancy in a proficiency test determined to be the
result of a systemic error (equipment, materials, environment) may be
handled according to Laboratory Quality Manual Sections 7.00
(Analytical Discrepancies) or 7.01 (Corrective Actions). This may
require a thorough review of log sheets for calibration, maintenance, or
critical reagent preparation. Once the cause has been identified, it
may be necessary to review relevant casework since the last successful
proficiency test by that analyst or method. Replacement equipment or
materials will be validated before being used for casework. The impact
of any environmental changes will be assessed before casework is
resumed.
9.5.4 Analytical/Interpretive Error
Any significant discrepancy in a proficiency test result determined to
be the consequence of an analytical or interpretive error will prohibit
the individual from further casework of that type until the cause has
been identified and corrected. The Laboratory Director will determine
the need to audit prior cases based upon the type of error and its cause.
Before resuming analysis or interpretation of case work, the analyst
must successfully complete an internal or external proficiency test.
9.6 Documentation
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The results of all proficiency tests will be maintained by the Serology section
in files located in the section. The proficiency test files will be maintained for
a period of five years. The Laboratory Director is responsible for maintaining
the Proficiency Test Logbook. The Laboratory Director is also responsible for
evaluating the Proficiency test results. He is to determine whether the
inclusions, exclusions and reported genotypes and/or phenotypes are correct
or incorrect.
Proficiency test results will be evaluated based upon section 8.2.2 in the STR
Procedure Manual.
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10. Audits
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
Audits are an important aspect of the QA program. They are an independent
review conducted to compare various aspects of the laboratory’s performance
with a standard for that performance. The audits are not punitive in nature
but are intended to provide management with an evaluation of the
laboratory’s performance in meeting its Quality Assurance policies and
objectives.
10.1 Frequency
Audits or inspections should be conducted annually by individuals
separate from and independent of the serology section. It is highly
desirable that at least one auditor be from outside the serology section.
10.2 Records
Records of each inspection should be maintained and should include
the date of the inspection, area inspected, name of the person
conducting the inspection, findings and problems, remedial actions
taken to resolve existing problems and schedule of next inspection.
The records are maintained in the Quality Manual, and in the green
file cabinet in the Serology section.
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11. Safety
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised:
All safety protocol and information is contained in a separate Safety Manual.
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12. Miscellaneous Reagents and Supplies
Effective Date: January 1, 2000
Initiated by: J. Burke, A. Harlukowicz
Approved by: Coroner
File: STRMAN 2003.DOC
Manual: STR Manual
Replaces: NA
Reviewed by: Quality Manager
Reviewed: Annually
Revised: January 16, 2003
The Serology/DNA section utilizes numerous reagents and supplies. Listed
across from each item below is the order number and company where each
item can be purchased. The supply companies listed below are merely
suggestions. If desired, alternate companies may be used.
Phenol/Chloroform/Isoamy alcohol
Microcons
2 ml Dolphin tubes
Spin –X insert w/o membrane
Quantiblot
Chemiluminescent detection reagents
X-ray film
Hybridization tray
Slot Blot apparatus
Gasket for Slot Blot
Biodyne B membrane
Sodium Chloride
Chelex Resin
Dithiothreitol
N-Lauroylsarcosine
Trizma base
EDTA
Sodium hydroxide
10N sodium hydroxide
Trisodium citrate, dihydrate
Citric acid, monohydrate
Proteinase K
Sodium dodecyl sulfate
Sodium phosphate, monobasic
Disodium EDTA
Potassium chloride
Potassium phosphate, monobasic
Anhydrous disodium phosphate
GeneScan 500 ROX Size Standard
310 Genetic Analyzer Buffer w/EDTA
POP4 Polymer
310 Capillaries
Sigma P-3803
Millipore 42413
Fisher 07200210
Fisher NC9217172
PE N808-0114
Pierce 34075
Amersham RPN.2103
PE N808-0136
Gibco BRL 1055AA, 1055AE
Gibco BRL 11055-084
VWR 28150-276
Sigma S-3014
Bio-RAD 143-2832
Sigma D-9779
Sigma L-5125
Sigma S-8524
Sigma E-5134
Sigma S-5881
VWR JT5674-3
Sigma C-8532
Sigma C-1909
Sigma P-4914
Gibco BRL 15525-025
Sigma S-3139
Sigma E-4884
Sigma P-3911
Sigma P-0662
Sigma S-3264
PE 401734
PE402824
PE 402838
PE 402839
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0.5 ml Genetic Analyzer Tubes
Septa for Genetic Analyzer Tubes
310 Glass Syringe
Profiler Plus/Cofiler Kits
Formamide
Dye Primer Matrix Standards
Microamp Reaction Tubes
PE 401957
PE 401956
PE 4304471
PE 4305979
PE 4311320
PE 401114
PE N801-0540
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