MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
PRINCIPLE:
This procedure uses the Perkin Elmer AmpFlSTR Profiler Plus and AmpFlSTR COfiler PCR
Amplification and Typing Kits. These PCR-dependent kits use Short Tandem Repeat (STR)
typing technology that detects length polymorphisms. Profiler Plus detects the gender marker
Amelogenin and the following nine STR loci: D3S1358, D5S818, D13S317, D7S820, D8S1179,
D18S51, D21S11, FGA, and vWA. COfiler detects the gender marker Amelogenin and the
following six STR loci: D3S1358, D7S820, D16S539, CSF1PO, TH01, and TPOX. When
Profiler Plus and COfiler kits are combined, all thirteen CODIS core STR loci are amplified with
two amplifications with two overlapping loci (D3S1358 and D7S820).
The Perkin Elmer GeneScan and Genotyper software are used to analyze data. The loci analyzed
are characterized in the table below. Three overlapping color spectra, plus a fourth red (ROX)
internal lane size standard, are automatically sized by GeneScan, and the corresponding base pair
(bp) alleles for each locus are called by Genotyper.
Locus
Designation
Kit
Chromosome
Repeat
No. of Common
Alleles
(in ladder)1
Alleles
Size Range
(bp)2
Dye Label
Blue:
3
TCTA (TCTG)1-3 (TCTA)n
8
12-19
114-142
5-FAM
vWA
Plus
COfiler
Plus
12
11
11-21
157-197
5-FAM
FGA
Plus
4
14
18-30
219-267
5-FAM
COfiler
16
TCTA (TCTG)3-4 (TCTA)n
(TTTC)3 TTTT TTCT (CTTT)n
CTCC (TTCC)2
(AGAT)n
9
5-15
234-274
5-FAM
X, Y
-
2
X, Y
107, 113
JOE
11
2
5
8
(AATG)n
7
8
10
12
5-10
6-13
6-15
8-19
24.238
169-189
218-242
281-317
128-168
JOE
JOE
JOE
JOE
189-243
JOE
21
9-26
273-341
JOE
D3S1358
D16S539
Green:
TH01
TPOX
CSF1PO
D8S1179
Plus
COfiler
COfiler
COfiler
COfiler
Plus
D21S11
Plus
21
Plus
18
Plus
Plus
Plus
COfiler
5
13
(AGAT)n
(GATA)n
10
8
7-16
8-15
135-171
206-234
NED
NED
7
(GATA)n
10
6-15
258-294
NED
Amelogenin
D18S51
Yellow:
D5S818
D13S317
D7S820
(AATG)n
(AGAT)n
(TCTR)n3
(TCTA)n(TCTG)n[(TCTA)3
TA(TCTA)3TCA(TCTA)2TCCA
TA](TCTA)n
(AGAA)n
22
1
There are a number of known rare alleles for most of the loci. The number given is the number of alleles in the
allelic ladder provided with the kit.
2
The size range is the actual base pair size of sequenced alleles contained in the AmpFlSTR Allelic ladder. The
sizes in the table include the 3’ A nucleotide addition. The size generated by the 310 Genetic Analyzer is usually
1-2 bp smaller.
3
R can represent either an A or G nucleotide.
1
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
SPECIMEN:
Samples amplified with the Perkin Elmer AmpFlSTR Profiler Plus and AmpFlSTR COfiler PCR
Amplification and Typing Kits and electrophoresed on an ABI Prism 310 Genetic Analyzer.
INSTRUMENTATION AND EQUIPMENT:
Apple Macintosh Power Mac computer
Mac-compatible Color Printer
GeneScan Software v 3.1, or greater
Genotyper Software v 2.0, or greater
QUALITY ASSURANCE:
1. Two qualified, proficiency-tested scientists must verify results.
2. Any papers taken from a post-amplification room should never be taken into a preamplification room.
3. Do not move or rename folders on the computers attached to the 310 Genetic Analyzer or it
will cause errors in the Collection and Analysis programs.
SAFETY:
Prolonged computer analysis may cause muscle soreness, eye fatigue, or other discomforts. No
employee shall be required to work more than two continuous hours on a video display terminal
(VDT). If greater than two hours are required, then the employee shall perform other work for
thirty minutes after each two-hour period on the VDT.
NOMENCLATURE:
1. Alleles are designated by a number, which presumably corresponds to the number of
tandemly repeated segments within that allele. An individual with a D3S1358-(14,16) profile
possesses one D3S1358 allele with 14 repeats and a second allele with 16 repeats.
2. Common variants exist in the Profiler Plus and COfiler systems. The most common variant
is a TH01 9.3 allele. In this case one base pair deletion is present in the 10th repeat. This
variant is more common than the “true” 10 allele. The loci that have variant alleles in the
ladder are D3S1358, vWA, FGA, D21S11, D18S51, and TH01.
2
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
PROCEDURE:
AUTOMATED ANALYSIS
This section of the protocol describes how to save, download, and analyze data from the 310
Genetic analyzer. GeneScan version 3.1 or greater software automatically analyzes each sample
as it finishes electrophoresing. Genotyper version 2.0 or greater software then converts allele
base pair sizes called in the GeneScan Analysis software into allele designations. The
AmpFlSTR Profiler Plus and COfiler Genotyper templates contain macros that find the allelic
ladders, create allele size categories (or bins) based on the ladder, assign the appropriate allele
label and peak height to samples, and filter labels from stutter peaks if below the predetermined
threshold level. Genotyper can also build tables containing the sample genotypes for printing or
importing into other programs.
DOWNLOADING SAMPLE FILES FROM ABI PRISM 310 GENETIC ANALYZER
1. From the Macintosh in the Analysis Room, double-click on the “310 #1 Run Folder” or “310
#2 Run Folder” icon to access the computer’s hard drive.
2. Locate the run of interest in the “Runs” folder within the “ABI Prism 310” folder. Drag the
folder to the “Casework Runs” folder on the “Forensic DNA Section” computer’s desktop.
The folder and its contents are downloaded to the hard drive.
3. Periodically, these files are downloaded and archived onto the CODIS Server and burned to
CDs (which are stored at the Crime Laboratory as well as off-site).
IMPORTING ABI PRISM 310 GENETIC ANALYZER SAMPLES
4. Start the Genotyper program by clicking on the “Apple Menu” in the upper left hand corner,
highlight the appropriate template, e.g. “MSP AmpFlSTR COfiler v3,” and release the mouse
button to launch the program.
5. From the File menu, choose “Import GeneScan File(s)...” Find and open the run of interest in
the Import dialog box. Ensure all four ‘Import colors’ boxes are checked, uncheck the
‘Import raw data’ box, and click “Import All.” Every sample file within the folder will be
imported into Genotyper. Alternatively, if the lanes of interest are in a GeneScan Project,
you may click on the project’s name and click “Import.”
6. The names of the imported samples can be viewed in the top pane of the Main window or by
clicking on the Views menu and choosing “Show dye/lanes window”. Highlight any
unwanted lanes (use shift-click to select multiple lanes), click the Edit menu and choose
“Clear” or “Cut” to remove extraneous lanes.
3
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
CHECKING INTERNAL LANE SIZE STANDARDS
7. Double click on the “Check GS-500” macro in the macro window located in the lower left
corner of the main window. The Plot window will open, showing the red electropherogram
traces for each lane, each peak labeled with its calculated size in bp.
8. It is easiest to print the GS-500 standard at this time (see “Printing Electropherograms and
Tables” below). First save the analysis results by clicking on “File” and choosing “Save as.”
Name the Genotyper project with a name that includes: case number, date of run, scientist’s
initials, and multiplex system. Save the data in the run folder from which the lanes were
imported.
9. Ensure each gs-500 peak within each sample is assigned the correct size, i.e. 75, 100, 139,
150, 160, 200, (~245), 300, 340, 350, and 400 bp. The 139, 150 and 160 bp peaks appear as
a tight group of three peaks and can serve as a point of reference.
10. The peak of approximately 245 bp is not sized to a preset value. Rather, it is used as an
indicator of run precision. The base pair size of this peak must be within a 1.0 bp window for
all injections used in a particular run.
NOTE: If there is an outlier, i.e. a sample whose 245 bp peak is outside of the 1.0 bp window for
a set of injections, this sample must be re-injected.
CALLING ALLELES
11. Double-click the “Kazam” macro in the macro window. The program will use the first lane
labeled “Ladder” to create a standard allelic ladder for each locus. “Kazam” then performs
the following calculations automatically:
a. Peaks greater than or equal to 150 RFU are labeled with allele designations or numbers
(because only peaks with a height greater than 150 RFU are sized in GeneScan). If the
peak height is greater than 8100 RFU from the raw data of GeneScan Analysis software,
then it is automatically noted in the “saturation” column of the 6-peak table.
b. Allele numbers are assigned to peaks that match the bp size of the allelic ladders (0.5
bp) of the corresponding dye color. Peaks that fall outside the 0.5 bp window of allele
sizes or are outside of the ladder’s range are labeled “OL allele” (Off-Ladder allele). If a
sample allele peak is labeled “OL allele”, then the sample must be reinjected to verify the
4
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
result. If reinjecting reproduces the off-ladder allele, the sample must be reamplified to
confirm the allele call.
c. Stutter peaks are typically four bases shorter (n - 4) or, in rare instances, 4 bases longer (n
+ 4) than the corresponding main allele peak. The proportion of the stutter product
relative to the main allele (percent stutter) has been measured for each locus by the
manufacturer and our validation study. The “Kazam” macro automatically filters out all
n-4 peaks that are less than the stutter threshold (listed in the table on the next page) of
the corresponding main allele peak.
BLUE
LOCUS
DESIGNATION
GREEN
STUTTER
THRESHOLD
D3S1358
vWA
FGA
D16S539
15
15
15
15
LOCUS
DESIGNATION
YELLOW
STUTTER
THRESHOLD
Amelogenin
TH01
TPOX
CSF1PO
3
10
10
10
D8S1179
12
D21S11
15
D18S51
18
LOCUS
DESIGNATION
STUTTER
THRESHOLD
D5S818
D13S317
D7S820
12
12
12
12. Once “Kazam” is completed, the Plot window will open, showing the blue electropherogram
for the first ladder in the upper pane and the blue electropherograms for sample lanes in the
lower pane. Each peak will be labeled with its corresponding allele number or as an “OL
allele” and the peak height in RFU. To inspect peaks for other colors, click on the
corresponding letter in the upper left hand corner of the main window (B=blue, G=green,
Y=yellow, R=red).
NOTE: Do not perform any editing of the electropherogram or table data on the computer. All
editing and note taking must be completed directly on the printed data.
13. Examine the positive amplification control (9947A) to ensure amplification has been
successful for each STR locus examined. Verify that the allele calls match the expected
allele calls for that locus. See “Expected Control Values” section at the end of this protocol
for correct values and what may cause deviations from the expected results.
5
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
14. Examine the negative amplification control to ensure that the amplification cocktail did not
contain any trace of genetic material. See “Expected Control Values” section at the end of
this protocol for correct values and what may cause deviations from the expected results.
15. Examine the reagent blank to ensure that the extraction solutions did not contain any trace of
genetic material. See “Expected Control Values” section at the end of this protocol for
correct values and what may cause deviations from the expected results.
16. A table of samples and their alleles may be created by double-clicking on “Make Table - 6
Peaks” in the macro window.
17. Save the analysis results (if not done earlier) by clicking on “File” and choosing “Save as.”
Name the Genotyper project with a name that includes the case number, date of run, and
scientist’s initials.
18. The project, corresponding lanes, and Genotyper template are periodically backed up on a
removable Zip disk, writable CD and/or network drives.
PRINTING ELECTROPHEROGRAMS AND TABLES
19. Before printing, be sure the Genotyper project is saved with a name that includes the case
number, date of run, and scientist’s initials since this prints on each page of the printout.
20. It is most convenient to print the red GS-500 data as soon as the “Check GS-500” macro has
been run (the red lanes are automatically displayed); these red peaks will not be labeled after
the “Kazam” macro has been run, so the “Check GS-500” macro will have to be run again).
21. To print the blue, green, and yellow (black) allele calls for each injection, click on the
corresponding letter in the upper left hand corner of the main window (B=blue, G=green,
Y=yellow, R=red) and then click on the plot window.
22. Click “Okay” to verify the title/header of the printout (the name under which the project was
saved) and “Okay” to accept the color printer.
23. To print a table, click on “View” menu and highlight “show table window” to make the Table
the active window. Change “Page Setup” to 65% print size and landscape to fit the table on
the page.
6
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
INTERPRETATION OF DNA PROFILES
This section of the protocol serves as a general guideline for the interpretation of STR profiles
when using the AmpFlSTR kits. However, it is not an exhaustive list of all casework scenarios.
Therefore, scientist experience and discretion are always taken into account before reporting STR
profiles. A second, qualified DNA analyst will technically review all data interpretations and
allele calls. If the analyst and the technical reviewer cannot agree on an allele call or data
interpretation, the technical leader will be conferred with to make the final call.
1. Peak identification.

Only allele peaks (Blue, Green, and Yellow) with a height >150 RFU can be reported, but
peaks below 150 RFU can help to assess the quality of a DNA profile and determine if
peaks are true peaks or artifacts, and if samples need to be reinjected (for 2, 5 or 10
seconds) or possibly reamplified.

The internal lane size standard (Red) is interpretable down to 50 RFU.
2. Assessing internal lane standard

Ensure that every sample injected and electrophoresed correctly by examining the internal
lane standards (red or ROX lanes). Clear, sharp internal lane standards peaks should be
present in all injections and be sized correctly (see above).

The smallest and the largest of the “245 base pair” peaks in a run should differ by no
more than 1.0 base pair. If there are outliers (peaks outside of the 1 base pair range),
those lanes need to be assessed. If there are true alleles present (that need to be sized), the
sample should be reinjected. If no alleles are present (such as in a reagent blank, negative
control, or sample with insufficient DNA to obtain a profile), the sample does not need to
be reinjected
3. True Allele Peak

An allele has a peak height greater than 150 RFU and typically less than 8100 RFU in the
raw data of GeneScan Analysis software (see note below regarding off-scale data), a
fragment size that falls within the base pair range, and has the appropriate dye color for
the loci. However, all peaks that have a peak height greater than 150 RFU are not
automatically typed as an allele. Stutter peaks, pull-up peaks, and background noise
could fall above 150 RFU, especially if the allele peaks are high. Peaks less than 150
7
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
RFU will not be reported, but should be evaluated in the interpretation of data. For these
reasons, the scientist’s experience is the most important factor in reporting alleles.
NOTE: Off-Scale Data: if too much DNA is added to the PCR reaction, the
fluorescence intensity from the PCR products may exceed the linear dynamic range
for detection by the instrument. Samples with off-scale peaks can exhibit raised
baseline, excessive pull-up of one or more colors, and/or high stutter peaks. The
peaks may even appear to have “flat tops” instead of sharp peaks. Samples with
off-scale data may be diluted and rerun or reinjected for shorter times.

Detection of off-ladder alleles. There are several rare alleles for which bins do not exist
in the allelic ladder. Rare alleles may contain 4 bp repeat units or repeat units less than 4
bp. If a rare allele is encountered, adding or subtracting the appropriate repeat length (1
bp if within the ladder, 4 bp if outside the ladder) from the closest allele in the ladder to
create a theoretical bin. The rare allele of interest must fall within 0.5 bp of the bin. If
the peak meets these criteria, the sample must be reamplified to confirm before reporting.
In order to declare a match, the same rare allele must be observed in both the questioned
sample and the relevant known sample.

Typically, within each locus, a peak balance of greater than or equal to 70% between two
peaks serves as an indicator of high quality, single source DNA producing a strong signal.
A peak balance of less than 70% should be interpreted with caution, as this is indicative
of degradation, the presence of a mixture, or low signal strength. Rarely, a profile with an
imbalance of true heterozygous alleles at a particular locus (which is reproducible even in
the known reference sample) has been documented.
4. Stutter peaks (“n – 4” and “n + 4”)

The most common stutter peaks are n - 4 peaks that have a fragment length four base
pairs shorter than the true allele. If the peak height of the n - 4 peak is less than the stutter
threshold expected for that locus, the peak is considered a stutter peak and is filtered out
by “Kazam.” However, if the peak height of the n - 4 peak is greater than the expected
stutter threshold, it may indicate a mixture (see below). If the sample is a known (single
source) and stutter peak is greater than the stutter threshold, then too much signal may be
detected (and signal strength is in non-linear range). The sample may be reinjected for
8
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
less time (2 seconds as opposed to 5 seconds) or reamplified with less DNA to aid
interpretation.

An n + 4 peak has a fragment length four base pairs longer than the true allele. N + 4
peaks are rare, but do occasionally occur. When evaluating an n + 4 peak the possibility
of a mixed sample must be explored. If the sample is a known (single source) and an n +
4 peak is present, then the sample may have too much DNA. The sample may be
reinjected for less time (2 seconds as opposed to 5 seconds) or reamplified with less DNA
to aid interpretation.
5. “n-1” peaks
AmpFlSTR kits are optimized to add an extra adenosine nucleotide (“A”) to the 3/ end of
the PCR product. When the reaction fails to add this “A” nucleotide to a significant
number of PCR copies, a peak one base pair shorter than the true allele may be observed.
This usually occurs because the amount of input DNA is too great, so the sample may be
reinjected for less time (2 seconds as opposed to 5 seconds) or reamplified with less DNA
to aid interpretation.
6. Pull-up Peaks
If too much signal is detected, an overlap in the emission spectra of the dyes causes a
DNA fragment to appear as multiple peaks in multiple colors. The pull-up peak should
have approximately the same fragment size as the true allele peak and can be
demonstrated by labeling all colors with scan number or base pair size. Reinjecting for
less time (2 seconds as opposed to 5 seconds) or reamplifying with less DNA may aid
interpretation.
7. Spurious Peaks/Anomalies
Spurious peaks are from artifacts or electronic noise and are not reproducible. If the
anomaly is in more than one color or outside of the allele calling range, interpretation can
be made from the one injection. If the anomaly is only present in one color and within
the allele calling range, the sample should be reinjected to demonstrate it is not
reproducible.
9
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
8. High Baseline/Background Noise
Low-level peaks that are generally less than 150 RFU, typically present in samples with
high signal. They are indistinguishable from the peaks present at or near baseline signal.
Analysts discretion should be exercised in interpretation. Rerunning the sample may aid
interpretation.
9. Highly Degraded Samples.

Highly degraded samples must be interpreted with caution: stutter peaks are generally
higher than those observed in high quality DNA; there may be spurious peaks from
degradation products; and there is a greater amount of unbalanced heterozygous alleles
(typically, the smaller allele will be no less than 50% of the height of the larger allele).

Trends to keeep in mind when analyzing Profiler Plus data in degraded samples: the
larger “FGA” locus had higher peak heights than the other blue loci; normally, the largest
loci are the first to demonstrate decreased peak heights as samples become more
degraded, but the smaller vWA locus may show lower signal; the smallest green locus
(D8S1179) may be present at the lowest levels of the three green loci; but the yellow loci
exhibit typical decreasing peak heights in larger fragments.

Trends to keeep in mind when analyzing COfiler data in degraded samples: amelogenin
and D3S1358 amplified robustly; D16S539 produced a good signal (albeit usually lower
than D3S1358) most of the time; and D16S539 may amplify even though CSF1PO may
be weak or nonexistent.
10. Inhibited Samples.
Inhibited samples are those samples that contain an impurity that inhibits the polymerase
enzyme activity or primer annealing. Inhibited samples may show differences in stutter
peaks, off-ladder alleles, allele or locus drop out, and unbalanced heterozygous alleles.
Inhibited samples differ from degraded samples in that the drop out of loci is not related
to base pair size of the loci. The hallmark sign of inhibition is the inability to obtain a
DNA profile even though DNA has been detected in the quantification procedure.
10
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
INTERPRETATION OF POTENTIALLY MIXED SAMPLES
The detection of more than two alleles per locus indicates a possible mixed sample. Variation in
peak height between alleles in a single locus may assist in the interpretation of such results. A
peak height balance of less than 50% could indicate a possible mixed sample or may be due to
unbalanced peaks at signal strength close to 150 RFU. Re-amplification of samples or increasing
injection time of samples may prove helpful by increasing peak heights of minor component
alleles. Allele peaks below 150 RFU should be considered to help in the interpretation of the
mixed DNA samples, but cannot be reported. The interpretation of mixtures should take into
account the possible confusion between a true mixture and the presence of stutter peaks. Be
aware of the potential of interpreting a mixed sample of two homozygotes as a single
heterozygote.
RECOGNITION OF MIXTURES
1. The detection of three or more alleles at two or more loci indicates a mixed sample.
2. Stutter greater than the threshold may indicate a mixture. Additional information provided
by other loci is necessary to confirm the presence of a mixture.
3. Heterozygous imbalance less than the threshold of 70% may indicate a mixture. Additional
information provided by other loci may be of assistance in these cases.
SEPARATION OF MAJOR AND MINOR CONTRIBUTORS
1. In a mixed sample of two apparent contributors at roughly equimolar concentrations, the
mixture either “excludes” or “cannot exclude” potential donors. Individual profiles cannot be
separated from the mixture, nor can random match probabilities be calculated.
2. In a mixed sample of two apparent contributors where the minor alleles are present at less
than 50% of the peak height of the major contributor (locus by locus), the major contributor’s
profile can be separated and random match probabilities can be calculated.
3. In a mixed sample of two apparent contributors where the minor alleles are present at less
than 50% of the peak height of the major contributor (locus by locus), the minor contributor’s
profile can be separated but random match probabilities can only be calculated for loci with 2
minor alleles (to eliminate the possibility of major and minor alleles overlapping).
4. In a mixed sample of three or more potential contributors (5 or more alleles at a locus), major
and minor contributors cannot be separated and random match probabilities cannot be
calculated. The mixture can either “exclude” or “not exclude” potential donors.
11
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
DNA PROFILE MATCH CRITERIA
THE FOLLOWING CONSTITUTE AN INCLUSION OR MATCH:
1. When making a direct comparison between two samples (e.g. a comparison between a
known and a questioned sample, or between two questioned samples), a match or
inclusion may be reported if there are no differences between two profiles at the loci for
which allele calls are determined.
2. A comparison may be made between the questioned specimen and other biologically
related family members (e.g. mother, father, and /or children) if a direct comparison is not
possible. An inclusion may be reported in the following paternity/maternity situations:
a. When comparing the DNA profiles of parent-child relationships, the questioned
sample and the reference sample must share at least one allele at every locus.
b. When comparing the DNA profiles of both parents to a child, the child’s profile must
possess one allele from each parent at every locus.
c. When comparing the DNA profile of a questioned sample to that of a spouse and
offspring, the spouse’s DNA profile should be subtracted from the profile of the
offspring. All remaining alleles within the offspring’s profile must be represented
within the questioned specimen’s DNA profile.
THE FOLLOWING CONSTITUTE AN EXCLUSION:
3. When making a direct comparison between two samples (e.g. a comparison between a
known and a questioned specimen, or between two questioned samples), an exclusion
may be reported where there is a single difference between the two profiles.
4. When comparing the DNA profiles of parent-child relationships, an exclusion may be
reported when two or more alleles present in the questioned specimen are not present in
the reference sample.
THE FOLLOWING CONSTITUTE AN INCONCLUSIVE:
5. When comparing the DNA profiles of parent-child relationships, a single allele difference
may be the result of a mutational event and therefore the final results are inconclusive.
12
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
THE FOLLOWING CONSTITUTE UNINTERPRETABLE:
6. When the peak heights are below 150 RFU for one or both of the samples compared, the
results are uninterpretable at that locus.
7. A mixture of three or more people may be reported as uninterpretable.
8. When a partial profile is obtained and reported, the statement “No results were obtained
at the remaining loci tested” shall be included in the report.
EXPECTED CONTROL VALUES:
1. Positive PCR Control (DNA 9947A):

The profile for the Positive control should be as follows:
D3S1358 = 14, 15
vWA = 17, 18
FGA = 23, 24
D16S539 = 11, 12
TH01 = 8, 9.3
TPOX = 8,8
CSF1PO = 10, 12
Amelogenin = X, X
D8S1179 = 13, 13
D21S11 = 30, 30
D18S51 = 15, 19
D5S818 = 11, 11
D13S317 = 11, 11
D7S820 = 10, 11

If the positive control does not work or does not type correctly, repeat the injection.

If the positive control does not work or types incorrectly repeatedly, the test results for
that set of amplifications will be rendered inconclusive and need to be reamplified.

Possible explanations for an incorrect or failed positive control include faulty control
DNA, carry-over of amplification product, contamination, failure to add control DNA or
instrument failure.
2. Negative PCR Control:

There should be no DNA profile in the Negative PCR Control.

If the Negative PCR Control gives an interpretable STR profile repeatedly, the test results
for that set of amplifications will be rendered inconclusive and need to be reamplified.
3. Reagent Blank Control:

There should be no DNA profile in the Reagent Blank Control.

If the Reagent Blank Control gives an interpretable STR profile, the Reagent Blank
Control should be reinjected. If the interpretable STR profile is reproduced, the test
results for the samples extracted with that particular reagent blank will be rendered
inconclusive. If sample size permits, DNA can be freshly extracted from the pertinent
13
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
test samples with a new Reagent Blank Control. The lots of reagents used should be
considered potentially contaminated and QC tested as soon as possible.
STATISTICAL ANALYSIS
Follow the SOP “Genetic Analysis” to determine the statistical significance of a match.
REFERENCES:
ABI Prism™ 310 Genetic Analyzer User’s Manual, Rev. 1, Perkin Elmer Corp., July 1995.
ABI Prism™ Genotyper® 2.0 User’s Manual, Perkin Elmer Corp., 1996.
ABI Prism™ GeneScan® Analysis 2.1 User’s Manual, Perkin Elmer Corp., September 1996.
AmpFlSTR Profiler™ PCR Amplification User’s Manual, Ver. A, Perkin Elmer Corp., 1997.
Profiler, Profiler Plus, COfiler Validation Folders, Forensic DNA Section, Maine State Police
Crime Laboratory.
Wallin, J.M., et. al., TWGDAM Validation of the AmpFlSTR Blue PCR Amplification Kit for
Forensic Casework Analysis, J. Forensic Sci., 1998, 43(4):117.
14
Version 2.1 3/13/03
MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
STR ANALYSIS AND INTERPRETATION
Reviewed
Date
Supervisor, Forensic DNA Section
Adopted
Date
Director, Crime Laboratory
Annual Review:
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
Reviewed
Date
15
Version 2.1 3/13/03