Forensic DNA Analysis
Introduction
Genetic information is carried in the form of DNA in all cellular organisms. DNA is the genetic
code that imparts our individuality. As the field of molecular biology has developed over the
last decade, so has its application to forensic identification and individualization. Increasing
knowledge of the human genome and improved detection technologies and the development
of the polymerase chain reaction (PCR) technique have increased the sensitivity, speed and
discrimination potential of DNA profiling.
Examination of DNA sequences unique to any given species can allow us to identify any type of
organism. Identifying individuals within a population is only slightly less precise.
For identification of an individual, we can examine specific regions of DNA as they vary from
person to person, in order to create a DNA profile or fingerprint of the individual in question.
There is an extremely low probability that another person has the same DNA profile for the
given set of regions.
DNA as a tool for forensic identification has broad applications, including:
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identification of potential suspects or subjects involved in a crime or who may have
contributed to a crime scene stain
exoneration of wrongly accused persons
identifying family relationships or establishing paternity
identifying victims of crime, war, catastrophe or other death investigations
Objectives
At the end of this course students should:
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Understand the principles of Forensic DNA profiling
Have an understanding of the range of profiling methods
Know the method of sample processing and sample limitations
Understand the significance of DNA profiling results and the limitations of the data
Know the methods used in DNA extraction and Quantitation
Know the components of a Genetic analyzer
Be familiar with DNA databases available and their applications
Understand the applications of Mitochondrial DNA
Assignment
Please note: The assignment is listed below. Please submit your assignment using the
'Assignments' Tool/Link. You should consider the assignment questions as you read through the
module content.
Forensic DNA Analysis
Consider a heavily soiled sweatshirt recovered from a murder suspect’s basement. It is dry and
appropriately packaged when submitted to the laboratory. One sleeve appears to have been
completely soaked in blood. Other droplets of blood not much larger than a pencil point can be
found elsewhere on the sweatshirt. The DNA analyst chooses three samples - one from the
middle of the blood-soaked sleeve, one from the edge of the large bloodstain on that sleeve
and one of the smaller blood droplets from elsewhere on the sweatshirt. Complete STR results
are obtained from the blood droplet and the sample taken from the edge of the large
bloodstain, but not the sample from the center of the stain. Why?
Brief History of Forensic DNA Profiling
Studies of the human genome have revealed families of interrelated arrays of repetitive
sequences among the non-coding or “junk” DNA. The number of individual repeats in
these long tandem repeat tracts is extremely variable and follows Mendelian inheritance
patterns. By extracting DNA and digesting it with a restriction enzyme that cut the DNA
outside but not within the repeat block, the size of each block of block of repeats could
be determined by electrophoresis, and the term restriction fragment length
polymorphism (RFLP) was applied.
The first application of RFLP analysis was in the study of genetic diseases. Many RFLP
sequences were mapped to a specific location on a chromosome. The gene associated
with a genetic disease, for instance Huntington’s disease, could be located by studying
the transmission of many RFLP sequences through the generations of a family. By looking
for RFLP sequences that were inherited along with the genetic disease, it was possible to
associate the disease with the chromosomal location of that RFLP sequence, enabling
identification of the specific disease-causing gene and the related defect.
While the field of medical genetics developed further, forensic serology was utilizing
protein and enzyme genetic polymorphisms for the purpose of identification. The source
of a body fluid or tissue could be narrowed down by determining the ABO type or by
studying variants in a few enzymes separated by electrophoresis and visualized using an
enzymatic activity-based color reaction.
These genetic polymorphism tests were limited by several factors; the amount of
variation in enzyme proteins is limited by functional requirements. Only a handful of
detectable functional variants exist for any one enzyme, and the limited number of
variants makes individualization impossible. Under the best circumstances, a forensic
serologist could state that a combination of variants would be expected to occur in only
one in several hundred individuals. The chance that any match between an individual and
a forensic sample was merely coincidence could never be discounted. In addition, not all
protein markers are found in all tissues. Most of the markers of forensic interest were
located on blood cells and were therefore not useful in characterizing saliva, semen or
vaginal secretions. The ABO types were present in these body fluids, but only in
“secretors” who make up about 80% of the population.
Forensic DNA Analysis
The limited number of variants also made it possible for one type to mask another: The
presence of an individual’s body fluids may not be obvious in a mixture with blood or
fluids from an AB secretor. Finally, the fragile nature of proteins leads to loss of
enzymatic activity and detectability after short periods of time in adverse environmental
conditions.
Around 1985, Sir Alex Jeffreys was credited with the first practical forensic application of
human DNA analysis. Jeffries used the RFLP method to identify the semen donor in a
serial rape-homicide case in Britain. This first use of forensic DNA profiling is described in
Wambaugh's The Blooding.
In 1986, two private companies in the United States began offering commercial DNA
testing. Lifecodes and Cellmark developed RFLP methods using restriction enzymes Pst I
and Hinf I, respectively. Due to the use of different restriction enzymes, data generated
by the two companies could not be inter-compared. The FBI went online in 1988 with a
method using the restriction enzyme Hae III which became fairly standardized among
state and local forensic laboratories.
Restriction Fragment Length Polymorphism
Similar to the Jeffrey’s method, the RFLP method identifies variation in the number of
repeats in tandem repeat tracts. The individual repeats range from 50 to 80 base pairs in
length, and DNA fragments resulting from restriction enzyme digestion range from about
500 base pairs up. After size fragmentation by gel electrophoresis, DNA is transferred to a
nylon membrane by the Southern blot method. The immobilized DNA was sequentially
probed with labeled DNA sequences homologous to different variable number of tandem
repeat (VNTR) sequences. Radioactive or chemiluminescent reporter tags attached to the
probes enabled visualization of the hybridized probes on X-ray film. One visualization
technique involved Ethidium bromide, which could be digitized, but was a potent
carcinogen.
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RFLP alleles exhibit continuous variation. Due to the large amount of variation, the
discrimination potential of the method is very high. A typical reported frequency
would be in the range of one in several hundred million.
 Compared to other methods, RFLP is not sensitive. Twenty-five ng or more DNA is
required.
 RFLP is sensitive to degradation.
 RFLP is labor-intensive and not amenable to automation.
Polymarker + DQA1
As the RFLP technique was being optimized the Cetus Corporation was developing a PCRbased method known as DQ-alpha, which was able to detect sequence variants in a
functional gene, the human leukocyte antigen (HLA). In this method, a small DNA
Forensic DNA Analysis
fragment was amplified using primers complimentary to conserved regions outside the
variable section. The amplified DNA was then hybridized to immobilized probes
complimentary to each variant. Colorimetric detection based on an enzyme linked to one
of the primers was used to determine which variant was present. Later, 5 additional loci
were added to the DQ-alpha test to produce the more discriminating Polymarker plus
DQA1.
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The limited variation detected by the PM+DQA1 test limited the discrimination
potential to less than one in 10,000, leaving open the possibility of a
coincidental match.
 Based on PCR, the sensitivity greatly exceeded that of RFLP method. Generally 1-2
ng of human DNA was sufficient.
 The size of the amplified fragments is 242-245 bases, making the method useful
on degraded DNA that could not be visualized with RFLP.
 PM+DQA1 could be completed in less than a day, but was still labor intensive due
to the need to hybridize the probes and carry out the colorimetric detection.
Amplified Fragment Length Polymorphisms (AMPFLPs)
The use of AMPFLPs, variable number of tandem repeats ( VNTRs) containing amplified
alleles, was developed as a compromise between the discrimination potential of RFLP
and the sensitivity and speed of PCR-based DQalpha/Polymarker tests. Based on short
repeat tracts mostly associated with functional genes, AMPFLP regions were short
enough to amplify. STRs are analogous to AMPFLPs but with smaller repeat units.
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The discrimination potential of AMPFLP loci was still much less than RFLPs. More
variants were present in AMPFLPs than in the PM+DQA1 loci, although certain
alleles were very common.
 As a PCR-based method, sensitivity was in the 1-10 ng range.
 AMPFLPs ranged in size from 200 to 1000bp, small enough to be useful in
degraded DNA.
 Although amplification was fast, detection was laborious and required use of
polyacrylamide gels and silver staining. Several AMPFLP loci were applicable, but
it was necessary to run separate gels for each.
Clean Technique
Clean Technique is a method developed to assist analysts in their efforts to produce
contamination-free work. It is in many ways an adaptation of Sterile Technique used in
Forensic DNA Analysis
microbiology laboratories. This technique is employed by DNA testing laboratories to
minimize cross contamination and the integrity of the acquired results.
To minimize contamination:
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Surface areas where samples are processed should be cleaned with a fresh 10%
bleach solution or appropriate disinfectant cleaner.
All instruments used to process forensic samples (e.g., forceps, scissors,
scalpel/razor blades, bone cutting equipment, pipettors and metal probes) must
be cleaned.
Samples should always be placed on clean surfaces and in sterile tubes.
Waste that may contain amplified DNA should be disposed of with great care.
Contamination of extraction areas with amplified product is the most difficult
kind of contamination to eradicate after it has occurred.
Finally the foot traffic in the lab where analysis is being conducted should be
limited.
Sample Processing
Questioned samples should be processed separately from reference standards, by
completing the analysis of questioned samples at a different time in a different location
than reference standards. This will eliminate the possibility of cross-contamination or
sample switching.
Each sample should be processed individually. Put away one exhibit before opening the
next. Work with very small or dilute samples prior to opening large or concentrated
samples.
Prepare a manipulation or reagent blank for each group of samples processed and each
extraction method used. A blank not only monitors for contamination in reagents, but
also on implements, consumables and tools.
Use clean instruments and surface protection for each item.
Sample Size and Sampling Size
The preservation and conservation of evidence is important to forensic DNA quality
assurance. The most effective challenge to a DNA test is re-analysis by a second
independent laboratory. Before testing the DNA analyst must decide whether there is
sufficient sample for one or more tests. When the quantity of sample is sufficient, a
portion of the sample is set aside and preserved for possible future re-analysis.
Forensic DNA Analysis
Degraded DNA
In assessing evidence stains, analysts must consider how large a sample is required to
obtain DNA results as well as the probable condition of the DNA. If samples are thought
to contain partially degraded DNA, more sample may be needed to obtain useful results.
Environmental conditions before, during and after the deposition of a stain and even in
collection, packaging and storage greatly affect its quality. If a drop of blood falls on a
cloth and dries quickly, the DNA will be well-preserved. If the drop of blood falls on cloth,
which remains damp for any appreciable period of time, microorganisms may degrade
the DNA in white blood cells (remember red blood cells don’t contain DNA). The surface
upon which the body fluid is deposited is also important. The DNA in human body fluid
cells will be degraded rapidly on items (rich in microorganisms) such as:
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foliage
soil
carpeting
any warm and moist surface
Handling of the evidence by law enforcement and crime laboratory personnel can also
affect the quality of any DNA that may be present. Most agencies have evidence
submission manuals that include handling; the FBI Handbook can be found at the
following website: http://www.fbi.gov/about-us/lab/handbook-of-forensic-services-pdf
Potential Mixtures
Many forensic evidence specimens contain stains or material from more than one
person. While mixed DNA profiles can be interpreted, the characteristics of the mixture
and the component of interest must be considered. It is important to extract the DNA
from an appropriate representative sample of any stains or items.
As an example, say the subject in an aggravated assault discards his shirt as he runs from
the scene. He gets away that night but is arrested later in the week and denies being in
the area of the time of the assault. The shirt contains bloodstains presumably from the
victim. However, it is necessary not only to verify that the blood originated from the
victim, but that the shirt originated from the subject. The DNA analyst tests one of the
blood stains on the shirt, and that DNA profile matches the victim. He then tests samples
(cuttings or swabbings) from the armpit of the shirt and also from the collar. Both
samples give the profile of the shirt’s wearer.
Significance of the Evidence
The relevance of any evidence to a particular case depends on the close interactions
between the DNA analyst, the crime scene investigator, the case investigator and the
prosecutor.
Forensic DNA Analysis
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The crime scene investigator has a brief window of opportunity to collect
evidence that may be relevant to the crime being investigated. Often the
offense has recently occurred at the time of evidence collection, and few details
are known. As such, the investigator will collect a broad range of evidence
samples, some of which will be more important than others. The crime scene
investigator or responding officer must document unique information relating
to the state of the scene and evidence collected.
 After preliminary investigation, the case investigator may be able to provide
information about the offense, such as any prior relationship of the victims and
suspects, statements to police, medical reports, and how various evidentiary
items relate to the crime.
 The prosecutor brings knowledge of defense theories, pertinent case questions,
legal standards of proof and an overview of information about the case gleaned
from many different sources.
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The DNA analyst contributes knowledge of the capabilities and limitations of
various DNA testing methods. The analyst knows what items are most likely to
produce profiles and what interpretations might be made regarding those
profiles. The analyst can use DNA to determine the source of a biological
sample, but not when or under what circumstances it was deposited
It may be argued that DNA analysts should be blind to the facts of the case and simply
work and report on the evidence submitted. The most unbiased results can be obtained
this way. The equivalent would be for the crime scene investigator to collect evidence
without knowledge of the type of crime and the case detective to investigate the case
without knowledge of victims or suspects. Regardless, perhaps the most beneficial reason
for allowing the DNA analyst access to certain aspects of the case is in enhancing his/her
ability to select and test evidence in an order consistent with its likelihood to yield results
and its probative quality. Because stains are not always easy to see, crime scenarios may
help lead the analyst to the best area for testing and save time for the analyst,
contributor and courts. Open communication with the investigator can also help select
the most probative evidence for priority testing
The value in showing that a victim’s own blood is on his clothing or possessions might not
be immediately apparent. Where severe injury is incurred, it is safe to assume that the
victim’s blood is present.
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It is not uncommon to find blood from multiple individuals on the clothing of
violent perpetrators. Lacking evidence to connect the suspect to the injured
victim, the DNA analyst might perform an exhaustive analysis of blood on the
victim’s clothing or other items from the scene, as the suspect may have been
injured during the event.
 It is not uncommon for a perpetrator to injure him/herself during a knife attack,
particularly when one hand is used to control the victim while the other holds
Forensic DNA Analysis
the knife. Most physical struggles will result in the transfer of some trace DNA
evidence from subject to victim.
Choosing the Technique
Currently there are three main varieties of PCR-based forensic DNA typing. The major
technique used in most crime labs is the STR multiplex which relies on nuclear DNA and is
applicable to all nucleated specimen types capable of yielding at least one nanogram of
DNA. Samples might involve saliva, blood, semen, vaginal secretions, body tissues, spongy
bone, and growth phase hairs with roots or sheaths.
A common evidence type not amenable to nuclear STR testing is hair shafts. A hair root
may contain a tissue tag or nucleated cells that will give STR results, but not the shaft.
The only DNA test capable of providing results on this type of material is mitochondrial
testing. Implemented by the FBI in 1996, mtDNA testing is also the method of choice for
badly degraded samples such as old skeletal remains. As mtDNA is inherited through the
maternal line, care must be given to the choice of reference standard. Any maternally
related individual or the children of a female individual could serve as reference
standards.
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In a mixture of male and female DNA where the female component is expected to
greatly exceed the male component, Y-chromosome STRs are a technique of
choice. Based on Y-chromosome STR loci, the test can indicate the presence of a
male to corroborate a story or yield a profile of a specified male.
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This test is almost exclusively used on mixtures of male and female DNA. Where
semen does not contain spermatozoa or the mixture consists of male saliva and
female secretions, nuclear STRs can discern an interpretable mixture up to
about a 1:10 ratio of male to female DNA.
Comparison of Techniques
Forensic DNA Analysis
For more information on Y-STRs, see:
http://www.cstl.nist.gov/biotech/strbase/y_strs.htm
More about mtDNA testing can be found at: http://www.mitomap.org/
The US Department of Defense employs mtDNA typing through its Defense Prisoner of
War/Missing Personnel Office.
http://permanent.access.gpo.gov/websites/dodandmilitaryejournals/www.dtic.mil/dpm
o/family/dnatyping.htm
Organic Extraction
Once the sample is chosen the DNA must be extracted from the sample before
electrophoresis can be conducted.Organic extraction is appropriate for most forensic
specimens including:
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Blood & blood stains
Epithelial cells
Saliva
Vomit
Feces
Urine
Sweat
Forensic DNA Analysis
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Semen (see differential extraction method)
Hair
Tissue
Bone
Organic extraction uses a digest buffer containing sodium dodecyl sulfate (SDS) to
denature proteins and the serine proteinase, proteinase K (pro-K) to cleave the proteins
into smaller fragments. Pro-K digestion helps lyse the cells and solubulizes components
while denaturing the protein. The mixture is combined with buffered
Phenol/Chloroform/Isoamyl alcohol, providing an alkaline environment and precipitating
the RNA and protein, separating it from aqueous DNA. Phenol and chloroform solubilize
polysaccharides and lipids respectively, while isoamyl alcohol prevents the solution from
foaming. The DNA partitions into the aqueous layer while protein remains at the organicaqueous phase interface. The sample is washed, usually with Tris-EDTA buffer (TE), and
filtered to remove residual extraction reagents and contaminants.
The amount of sample used is determined based on the sample type, concentration,
substrate and expected condition.
Blood stains may be extracted directly from a cutting of the fabric on which they were
deposited except when found on blue denim. The dye in denim is a PCR inhibitor, so
swabbing a bloodstain on jeans is the best method of recovery. Calcium alginate swabs
are frequently used in the medical profession to collect cultures, but have also been
shown to inhibit PCR. Cotton swabs should be used for DNA extraction.
Chelex Extraction
This method is appropriate for many forensic specimens including:
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Blood & blood stains
Epithelial cells
Saliva
Hair
Semen (see differential extraction method)
Chelex extraction is harsh compared to proteinase digestion and organic techniques. This
should be considered when dealing with degraded or very old samples. The Chelex 100
Ion Exchange Resin is comprised of styrene divinylbenzene copolymers with paired
iminodiacetate ions acting as chelators. It is highly selective for heavy metals and divalent
cations versus monovalent cations. Produced by Bio-Rad Laboratories, Chelex 100 is
Forensic DNA Analysis
widely used in the forensic community. The resin is designed and certified for the
extraction of DNA ready for amplification and for complete removal of metal PCR
inhibitors. The basic nature of Chelex resin keeps extracted DNA in a single stranded state
available for concentration and/or quantitation.
Blood
Samples are first extracted in DI water or saline to help free cells from the substrate and
to lyse any red blood cells. The supernatant, which may contain heme, can be discarded.
Washing steps help remove any residual protein. Cells are then incubated with Chelex
and lysed by boiling.
Epithelial Cells
Chelex extraction is a rapid method for extraction of DNA from epithelial cells found on
items such as cigarette butts, envelope flaps, stamps, buccal swabs, beverage containers
and swabbings or cuttings from other textiles or hard surfaces. Most samples bearing
epithelial cells require concentration to obtain amounts applicable for quantitation and
amplification. Freshly smoked butts are an excellent source of DNA. Butts that have been
exposed to the environment may or may not have been subject to degradation. Used
chewing gum may give better results using organic extraction methods.
Hairs
Generally, hairs that just drop out are in telogen or resting phase and do not possess
nucleated root cells or a tissue tag and will not provide sufficient DNA for typing. Hairs in
the growth or cessation stages (anagen or catagen) may have nuclear DNA in their roots.
Hairs, forcibly removed, may or may not have tissue tags. It is very important to clean the
hair thoroughly to remove any foreign body fluids or other materials. Retain the hair
shaft as a control. Also, if nuclear DNA testing fails, the shaft may be sent for mtDNA
analysis. Approximately a 1cm length of hair, obtained form the root end is necessary for
analysis. Hairs mounted on slides can also be recovered for analysis.
Sperm
Mixtures containing spermatozoa provide a unique opportunity to separate the DNA
profiles of the contributors. This is achieved by isolating the sperm cells during extraction.
Sperm cells resist digestion by extraction buffer and proteinase K. By leaving out the DTT,
it is possible to release the DNA from other types of cells while leaving the sperm intact.
After pelleting and washing the sperm, a second extraction employing DTT lyses the
sperm cells to release their DNA.
Image courtesy of: HowStuffWorks: http://people.howstuffworks.com/dna-evidence.htm
Forensic DNA Analysis
Organic Differential Extraction
Organic extraction of stains or swabs either known or suspected to contain sperm calls
for a slightly altered procedure. The most commonly encountered example would be
vaginal swabs from a positive sexual assault case. First, the sample is extracted into
deionized water and the cell debris pellet evaluated microscopically for the presence of
epithelial and sperm cells. A second portion of the sample would then be required for the
actual differential extraction.
If epithelial cells are present, they are preferentially lysed by Proteinase K. Following
centrifugation; the supernatant containing the lysed epithelial cells is organically
extracted for the epithelial cell DNA. The remaining pellet, containing any sperm, is
washed and lysed by a combination of Proteinase K and dithiothreitol (DTT) and then
organically extracted. Timing is imperative in differential extraction as sperm cells may
lyse in the initial pro-K step and contaminate the epithelial cell DNA fraction.
Chelex Differential Extraction
As with organic differential extraction methods, this procedure allows for differential
extraction of DNA from stains containing sperm using Chelex resin. The sample is first
extracted using DI water and the pellet evaluated for epithelial cells and sperm, if
present, epithelial cells are preferentially lysed by Proteinase K. After centrifugation, the
supernatant containing the lysed epithelial cells is removed and mixed with 20% Chelex.
The remaining sperm pellet is washed, lysed using pro-K and DTT. This sperm digest is
then mixed with 5% Chelex. After boiling with Chelex resin to remove ions and inhibitors,
the two fractions of DNA are then ready to be concentrated and or quantitated.
DNA Extraction Kits
Several DNA extraction kits are commercially available. Most use either modified glass or
magnetic beads which in the presence of chaotropic salts promotes binding between
DNA and the specialized glass particles. Some kits are optimized for extraction from
specific substrates, such as blood or feces.
QIAmp Kits from Qiagen addresses whole and partitioned blood containing common
anticoagulants, bone marrow, feces, body fluids, and other stains of serological value.
The QIAmp silica-gel membrane binds nucleic acids as other contaminants pass through.
Inhibitors to amplification and proteins are removed in two wash steps, and the DNA is
eluted and ready for analysis.
Dynabeads®, CST® and BioMag® beads use silica coated magnetized beads that capture
leukocytes, shortening the extraction process. A series of wash steps and lysis leaves
nothing in sample tubes but DNA and beads.
Forensic DNA Analysis
Overall, these proprietary kits offer rapid extraction, but may require additional washing
in order to isolate samples appropriate for PCR. Cost-effectiveness is also a consideration
as is the use of methods already validated and accepted within the forensic community.
DNA IQ TM of the Promega Corporation is amenable to automation and extracts DNA at
set quantities. This saves time in the extraction of multiple samples containing significant
amounts of DNA. One disadvanatage of many of the proprietary systems is the failure of
the method to maintain the DNA in a single-stranded state, requiring additional steps of
heating the sample in lysis buffer prior to preparation for amplification.
Yield Gel
Yield gel quantitation of DNA with ethidium bromide visualization of DNA bands in an
electrophoretic submarine gel is used to assess the total amount of total DNA present
(from any source) and the relative size (condition) of the DNA fragments. This technique
helps to determine a ball-park range of DNA quantities, preventing under- or overloading
of the slot blot
For pristine specimens, un-decomposed soft tissue or liquid blood, the yield gel provides
a good estimate of total human DNA. The amount of non-nuclear DNA from mitochondria
is insignificant. For most other types of samples, oral swab reference standards, forensic
specimen blood and semen stains, and saliva-containing samples, the human component
of the total DNA present can vary considerably in both quantity and condition. The yield
gel should be considered a useful, yet preliminary tool in quantitation.
Slot Blot for Human DNA Quantitation
In a Slot Blot analysis to determine how much human DNA is present. Quantitation is
assessed via hybridization with a higher primate specific probe (D17Z1) complementary
to an alpha-satellite DNA sequence located on chromosome 17: An aliquot of extracted
DNA mixed with Spotting Solution is applied to a Biodyne B nylon membrane along with a
serial dilution of known human DNA standards. Spotting solution contains EDTA to
protect the DNA, bromothymol blue as a tracking dye for loading control and sodium
hydroxide to denature the DNA and activate the nylon membrane. The surface of the
Biodyne B membrane becomes positively charged at basic pH and binds the negatively
charged backbone of denatured DNA, permanently binding the DNA to the membrane.
The membrane bound DNA is hybridized with a biotinylated oligonucleotide probe,
D17Z1, attached to a reporter system that permits the quantitative visualization of the
DNA, which is complementary to an interspersed DNA sequence found only in humans
and higher primates. The amount of the biotinylated probe which hybridizes to DNA
bound in each oval-shaped slot is proportional to the amount of human DNA present. In
this technique, sample is transferred to the membrane though the use of a vacuum
applied to the blot apparatus. Bands form as the Bromothymol blue is deposited on the
membrane and the remainder of the solution is suctioned through. The blue color, TMB
Forensic DNA Analysis
or blue dye, is a loading control, eliminated before visualization. The limits of detection
are between 10 and 0.15 ng. Aliquots of DNA samples having DNA quantities outside of
this range cannot be accurately measured.
Fluorometric or Luminometric Assessment
DNA intercalators in the form of dyes or excitable enzymatic systems can be used to label
DNA for detection by spectrophotometers, fluoremeters or luminometers. Common
research protocols for sequencing, cloning, transcription, transfection and others all
benefit from defined template concentrations, as does preparation for STR amplification.
Forensic DNA analysis tends not to use this type of quantitation, as it is labor intensive
and not as reliable as other techniques.
Spectroscopic Methods
Once the DNA is isolated and extracted, the amount of recovered DNA can be estimated
using spectroscopic methods. Nucleic acids have UV-Vis absorption characteristics in the
240-280nm range due to the chemical nature of the purine and pyrimidine bases. This
absorption is influenced and modulated by the stereochemical and conformational
effects of the ribose-phosphate backbone. In this process sample readings are taken at
wavelengths of 260 nm (OD260) and 280 nm (OD280). This measures both nucleic acids
and protein present in the sample (260 nm for nucleic acids and 280nm for protein
concentrations). The ratio between the readings (OD260/OD280) provides an estimate of
sample purity. An OD of 1 in a 1 cm path length = 50μg/ml for double-stranded DNA; An
OD of 1 in a 1 cm path length = 40μg/ml for single-stranded DNA or RNA.
MALDI TOF
Matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDITOF-MS) has widespread applications as an analytical tool. With a mass range of 1300kDa, high accuracy, and sensitivity this technique is suitable for the quantitation and
analysis of nucleic acids.
Mass spectrometry of DNA is more complex than protein analysis, due to the acidic
nature of DNA, which leads to the formation of sodium and potassium adducts. The
combination of extensive washing procedures and the use of 3-hydroxy-picolinic acid as a
sample matrix has facilitated the development of this application. This technique is used
more for sequencing or STR analysis than for DNA quantitation.
Real Time PCR Method for Amplifiable Human DNA Quantitation
Forensic DNA Analysis
The ABI Prism 7900 Real Time Quantitative PCR Instrument (TaqMan®) can be used to
detect accumulation of PCR product and permit accurate quantitation in the exponential
phase of PCR reactions. Real time PCR is a well-established method of DNA quantitation
used by researchers for several years. From modification of the polymerase chain
reaction, RT PCR is carried out in a specialized thermal cycler that can detect the amount
of amplified DNA present. A very small aliquot of the extracted DNA is amplified, and
once the quantity of amplified DNA reaches a predetermined level, the amount of
starting DNA can be calculated. This system can handle concentrations of DNA ranging
from 0.003 to > than 50 ng/μL. Whereas the yield gel measures the amount and condition
of total DNA, and the slot blot measures the amount of isolated higher primate DNA, the
RT PCR system measures the amount of amplifiable human DNA.
Another version of the system measures amplifiable male human DNA, which together
with the original system, can help in choosing between the use of somatic or Y-based STR
multiplexes in cases involving mixtures of male and female DNA.
The quantity of DNA in test specimens can be assessed by comparison with DNA
standards. The standards and high molecular weight DNA will move as a band not far
from the origin. If the sample band is greater than the 128 ng standard or if there is DNA
remaining at the origin, the sample can be diluted and rerun.
DNA Amplification
Amplification of extracted and quantitated DNA samples is a process where DNA is
copied to generate sufficient sample for analysis. Amplification using PCR is a method of
chemically copying DNA. The polymerase chain reaction is carried out in a thermal cycler
under very specific conditions. PCR is applicable to a vast number of research, clinical and
forensic analyses. RT-PCR, Hot Start PCR, Long PCR, Real Time PCR, Single Locus PCR,
Differential PCR, are all in use, but the main focus in forensic DNA analysis is multiplex
PCR short tandem repeats.
Multiplex PCR calls for adding more than one set of PCR primers to the reaction, targeting
multiple locations. Appropriate for forensic DNA analysis as the probability of identical
alleles in two individuals decreases with an increase in the number of polymorphic loci
examined.
In optimizing a PCR reaction, the exact length and placement (therefore sequence) of the
primers, the annealing temperature, extension time, Mg 2+ concentration, enzyme
concentration and other factors must be optimized. The two primers must not have
sequences such that they can anneal to each other. Considering the many parameters
that must be harmonized in order to optimize a single PCR reaction, any slight deviation
from protocol or alteration of conditions or input DNA will lead to a failure to obtain
robust results.
Forensic DNA Analysis
There are two sources of forensic STR multiplex reagents, that market both one-shot
multiplex reactions, which amplify 13-16 loci simultaneously, as well as systems that
amplify fewer loci. These products have been thoroughly tested and validated to comply
with DNA Advisory Board standards. These are widely accepted in the forensic
community and data generated using both can be included in the FBI’s national CODIS
(combined DNA index system) database.
The 13 CODIS core STR loci include: TPOX, D3S1358, FGA, D5S818, CSF1P0, D7S820,
D8S1179, THO1, VWA, D13S317, D16S539, D18S51, D21S11. The gender marker
Amelogenin is also considered. These STRs are chosen for their regular repeat unit,
distinguishable alleles, robust amplification, and their high heterozygosity. Alleles are
generally named for their number of repeats; The D refers to DNA, the S versus Z
(example D17Z1) designation refers to whether that tandem repeat is single or multicopy
and the first number in the sequence to the chromosome one which the respective STR is
located. Because of the nature of this technique and the risk of sample contamination,
The Clean Technique principles of contamination prevention must be used at the
amplification reaction step. Roadblocks to optimum PCR product include: contamination,
too much or too little DNA, the presence of reaction inhibitors, and the quality of DNA in
the original sample.
For animated descriptions of PCR see the following websites:
http://www.bio.davidson.edu/courses/Immunology/Flash/RT_PCR.html
http://depts.washington.edu/genetics/courses/genet371b-aut99/PCR_contents.html
http://users.ugent.be/~avierstr/principles/pcr.html
The Genetic Analyzer
The most basic components of a genetic analyzer include: separation power supply, heat
source, sample trays, pumps, optics, a detection unit, and computer systems.
Consumables may include: tubes, buffer, polymer, capillaries, and critical reagents such
as size standards.
The underlying premise by which capillary electrophoresis operates is that electrical
species (negatively charged DNA) suspended in an electrolyte will migrate according to an
applied electrical current. DNA fragments should travel away from a negatively charged
electrode (cathode) and toward a positively charged electrode (anode). High voltage is
applied across a narrow bore capillary filled with electrolytic solution, achieving
separation. Molecules with different mass to charge ratios are readily separated.
Compared to gel electrophoresis and other separation methods, CE is easily automated
Forensic DNA Analysis
and reproducible, fast and with high separation efficiency, with a small sample size
requirement. Near the cathode end of the capillary, there is a photo-receptor and
detector light source. The typical mode of detection is fluorescence detection. An argon
ion laser excites the dyes. A charge-coupled device or CCD camera detector monitors 525
to 650nm fluorescent wavelengths. When light photons fall on the array of pixels, the
silicon of the CCD absorbs the energy and fosters a reaction to convert light into an
electronic charge. The number of electrons collected at each pixel depends on light level,
exposure time, and wavelength.
During the amplification process, STR regions are labeled with specific fluorescent dyes.
Samples containing the DNA are placed in a genetic analyzer (usually via an autosampler).
CE is used to separate the STR segments by size, a laser source measures their fluorescent
emissions.
A typical instrument consists of: autosampler, injection electrode, capillary, laser source,
CCD camera, syringe with polymer, pump block with specific ferrules, and containers for
both inlet and outlet buffer. Collection software is used to convey parameters to the
instrument such as: run temperature, injection time, injection order, injection voltage,
run voltage, run time, and others. This software is also used in maintenance of the
instrument and general operation. Modules, matrix files, and data storage are also
controlled using the same programs. The most commonly used genetic analyzers are a
product of Applied Biosystems, the ABI Prism® 310, 3100, 377 or 3700 instrument
system. Other manufacturers of instrumentation include Amersham Biosciences
(MegaBACE™ DNA Analysis Systems) and Beckman Coulter (CEQ™ Series).
Information collected by the CCD camera is directly translated into an electropherogram
which contains data such as the name of the locus, the number of repeats, size of the
fragment and peak height. Upon conversion of data regarding allele sizes in base pairs
into allele designations, genotypes are assigned by comparison with the appropriate
allelic ladders.
Databases
One of goals of CODIS (the FBI’s Combined DNA Index System) is the creation of a
database of states’ offenders’ profiles. This would aid in solving crimes where there are
no specific suspects. The national DNA identification index was authorized by the DNA
Identification Act of 1994 and has been in operation since October, 1998. Another
important aspect of CODIS is its use as a population statistics database, for research and
protocol development and for quality control if personally identifiable information is
removed.
Forensic DNA Analysis
Likelihood ratios and match probabilities are widely used statistical measurements used
in assessing or explaining the significance of DNA evidence. The impact these interpretive
statistical calculations have on a jury is immeasurable. While the burden of proof lies in
the hands of the prosecutor, it is debatable whether the prosecutor or appellant should
issue a warning to the jury regarding interpretation when the prosecution is relying upon
mathematical calculations and interpretation of statistical evidence.
Likelihood ratio (LR) tests are a powerful means of testing assumptions. It is the ratio of
the match probability if the forensic sample and the subject or suspect sample came from
the same person and the match probability that they came from different parties. Match
probability is another frequently used presentation of forensic calculations, calculated
from the frequencies of DNA markers in a database.
As of April 2003, all states and the US Army and FBI participated in National Data Index
System (NDIS) except for Mississippi and Rhode Island. In the US, some states hold
voluntary samples, often provided for exclusion in investigations, but the NDIS at the FBI
consists only of convicted offenders. CODIS is a coordinated system of local, state and
national databases, which enables crime labs to exchange and compare profiles
electronically. The CODIS homepage can be found at:
http://www.fbi.gov/about-us/lab/codis/codis_brochure
One Laboratory Information Management System originally developed for the South
African Police Services Forensic Laboratory includes a built-in database and includes
components called STRlab, which is made available for free to those involved in forensic
STR casework. In the year 2000, the United Kingdom announced that its database
reached 1 million profiles, which was estimated to be approximately one third of the
actual criminal population. The CrimTrac Agency was developed in Australia in 2000 with
substantial provisions for a National Criminal Investigation DNA Database. A later module
addresses paternity and identification.
The Significance of a Match
The Evaluation of Forensic DNA Evidence, is available online from the National Academies
Press at http://www.nap.edu/catalog/5141.html. Many labs employ software to aid in
appropriately crunching the numbers.
PCR-based analyses has facilitated typing of minute amounts of evidence, allowing for
more matches to be made between standards and crime scene evidence and in cold hits.
In theory, a profile match can aid in the prosecution of a criminal or exonerate the
wrongly accused. States and the federal government are constantly assessing the scope
of their convicted offender databases and crime laboratory practices. The expansion of
Forensic DNA Analysis
databases has proven their effectiveness. FDLE estimates that approximately 50% or
more of rapists in the state have prior convictions for burglary. Expanding the database to
include profiles of burglars is expected to have a major impact on the resolution of sexual
assault cases.
Occasionally or in the event a body is badly decomposed and parental lineage is
unavailable, secondary standards known to belong to the individual in question may be
used in casework, such as make-up applicators, toothbrushes, jewelry, and other
personal items that might hold traces of the person’s biological material. Probabilities
that the profile from the deceased match the profile from the material extracted from
the secondary standard can be calculated and support any other circumstantial or
physical evidence leading to an identification.
Reverse paternity techniques can be used to identify the contributor of a profile if one or
both parents or several other relatives such as siblings are available to provide standards.
Unlike parentage testing, which seeks to investigate whether the alleged individual is
truly the mother or father of a child, forensic identification in a missing or unknown
deceased person case seeks to identify the deceased by extrapolating from known
samples
Mitochondrial DNA
Mitochondrial DNA (mtDNA) is a useful tool in the event that available samples are
degraded and incapable of providing nuclear DNA for traditional forensic DNA analysis.
mtDNA is a circular DNA molecule found in the mitochondria of a cell. While only one
copy of nuclear DNA exists in the cell nucleus , a typical cell may have hundreds of
mitochondria. Mitochondria have some unique DNA and ribosomes, and can make many
of their own proteins.
Because they are present in the female gamete at conception, mtDNA is maternallylinked. In fact, 99.99% of mammalian mtDNA is inherited from the mother. The other sect
of mtDNA is incorporated as the sperm carries approximately 100 mitochondria on its tail
region as opposed to the ~100,000 found in the oocyte. By comparing regions of mtDNA
between a forensic unknown and the possible maternal relatives, one may be able to
support a relationship. Less power of discrimination exists compared to nDNA analysis, as
an mtDNA haplotype is not unique, but consistent throughout maternal lineage. Usually,
only a single haplotype is passed on to an offspring. The presence of more than one
haplotype in an organism is referred to as heteroplasmy. Obviously, some mutations exist
amongst and individual’s vast number of mitochondria, but only one haplotype is
typically identified. The overall advantage lies in the likelihood of obtaining a profile from
hair shafts, old or damaged bones, or samples that have been submerged. The offering of
mtDNA services varies amongst and between forensic laboratories.
Forensic DNA Analysis
Most forensic mtDNA testing is performed by sequencing of two hypervariable regions
designated HV1 (HVI) and HV2 (HVII). Because they contain the greatest variability among
maternally unrelated persons, HV1 and HV2 are generally considered suitable for
determinations of identity.
The US Armed Forces Institute of Pathology has outlined a DNA Outreach Program to
insure proper identification of fallen servicemen. Trial courts in some states have yet to
demonstrate admissibility of mtDNA evidence in criminal cases, but with increasing
technology and general awareness, it is expected that mtDNA analysis will rapidly join
nDNA analysis by PCR and RFLP or STR as an accepted tool in forensic identification.
Valuable information is provided by "MITOMAP: A Human Mitochondrial Genome
Database" 2003, available at http://www.mitomap.org.