1. COMPARISON OF
COMMERCIALLY AVAILABLE
STR TYPING KITS: SAMPLE
CONDITION, ACCURACY,
AND EFFICACY
By Courtney Brennan
Special thanks to my Mentors/Professors:
M. Guido, K. Sweder, and M. Sponsler

2. BACKGROUND
 Many labs use commercially available “kits” to type
DNA samples
 These are usually “STR” (short tandem repeat) typing
kits.
 Many labs only use one kit, and most forensic labs
only use one kit per sample.
 The two most common producers of these kits are
Applied Biosystems and Promega Corporation.
 Many kits include the 13 core loci (CODIS)
 A Loci is a specific location on a chromosome where
a sequence or gene is found
 Variants of this sequence are known as alleles

3. BACKGROUND (CONT.)
 Homozygotes have only
one form of an allele
CC
 Heterozygotes have
varying forms of an
allele
 These dif ferent
CT variations on the loci
are read and compared
in DNA profiles
 Amelogenin, the sex
determination loci, is
TT commonly analyzed in
these kits
http://carcin.oxfordjournals.org/content/25/8/1443/F1.expansion.html

4. UNDERSTANDING ACCURACY
While kits tend to be far within the acceptable
range of accuracy for typing, accuracy has
costs
Increased accuracy may decrease the real-life
application of the kit since contaminants or
mixed profiles would be more difficult to
analyze
Some samples with only a small amount of
DNA can exhibit inaccurate readings if they
have low copy number

5. ANALYSIS OF AMPFISTR® SGM PLUS™
MULTIPLEX SYSTEM FOR EFFECTIVENESS ON
LOW COPY NUMBER SAMPLES
AMPFISTR® SGM Plus™ system was tested for
usefulness in analyzing LCN (low copy
number) samples
Low copy number generally refers to samples
in which there is less than 100pg of input
genetic material
This approach can have issues – sometimes
one allele is not present in a large enough
amount to be detected properly

6. ALLELIC DROPOUT IN LCN CONDITIONS
Difference #
Allelic dropout (% of
Locus observed in 28 vs. Alleles
expected)
34 cycles
Amelogenin 6 [4H] 4 (14.8)
D3S1358 8 [3L, 2H] 5 (10.6)
HumVWF31 10 [5L, 2H] 7 (10.4)
D16S539 10 [4L, 4H] 8 (17)
D2S1338 13 [4L, 2H] 6 (8.9)
D8S1179 11 [2L, 2H] 6( 8.9)
D21S11 6 [4L, 2H] 4 (14.8)
D18S51 11 [4L, 2H] 6( 8.9)
D19S433 6 [4L, 2H] 6( 8.9)
HUMTHO1 1 [1H] 1 (3.7)
HumFIBRA 13 [2L, 4H] 5 (7.4)
• Allelic dropout is when one or more of the alleles present in a sample
are either poorly detected or undetected in a profile
• This results in false homozygosity – being led to believe a specific loci
is homozygous when in fact the analysis simply failed to pick up the
second allele

7. ANALYSIS OF AMPFISTR® SGM PLUS™ MULTIPLEX
SYSTEM FOR EFFECTIVENESS ON LOW COPY
NUMBER SAMPLES (CONT.)
The average allelic dropout rate among the
loci analyzed was, on average, around 7-14%
10% of the heterozygotes analyzed showed
allelic dropout
The author(s) found that while the AMPFISTR®
SGM Plus™ system was reasonably able to
analyze LCN samples, special consideration
should be taken when reading results from
this type of sample

8. USE OF APPLIED BIOSYSTEMS’ AMPFLSTR
PROFILER PLUS™ IN PERSONAL
IDENTIFICATION CASES
This study was examining the effects of
sample size on the ability of the AmpFlSTR
Profiler Plus™ to clearly detect various alleles
Measurement is in relative fluorescence units
(RFU): units of fluorescence intensity which
signify the “strength of DNA detection” so to
speak
Different sources were used for the same DNA
profile so that differing amounts of genetic
material were retrieved

9. DNA AMOUNTS AS MEASURED BY RFU
Heterozygous
DNA (ng) D3S1358[L/H] VWA[L/H] FGA[L/H] D18S51[L/H] D7S820[L/H]
4 1291/1181 1119/907 701/678 798/601 591/459
2 1005/927 773/693 593/538 633/519 486/390
1 655/695 664/435 499/441 597/575 340/407
0.5 385/341 225/212 229/218 197/226 173/155
0.25 132/169 127/101 104/106 114/66 63/77
0.125 78/107 75/68 56/59 55/39 41/45
0.063 51/42 45/47 37/38 38/31 28/31
0.031 47/41 - - 28/26 -
Homozygous
DNA (ng) X D8S1179 D21S11 D5S818 D13S317
4 2619 2221 2018 1789 1162
2 1608 1624 1309 1368 1013
1 1131 906 1000 1110 1004
0.5 738 446 440 484 431
0.25 343 262 177 244 204
0.125 180 136 122 112 108
0.063 95 71 61 62 58
0.031 40 29 47 25 35

10. THE IMPACT OF SAMPLE SIZE ON DETECTION
 When the sample size reduced to that of 0.015ng or less
of DNA, only two alleles were even detectable:
X
 D21S11
 Homozygous alleles were (as expected) easier to detect
 Using these numbers, it was determined that the optimal
sample size for DNA was around 1-2 ng of product for the
AmpFlSTR Profiler Plus™ kit
 The authors, Buscemi, et al., concluded that the success
rate in forensic casework samples seeking personal
identification was around 75% before purification
 This rate rose to 90% when samples that were not
originally amplifiable used the microcon -30 purification
unit

11. AMPFLSTR® IDENTIFILER® AND
MOTHERLESS PATERNITY TESTS
 A study was done to determine the effectiveness of
the AmpFLSTR® Identifiler® kit in cases where the
mother’s sample was not available for comparison in
paternity testing
 Paternity testing generally involves three samples;
this method was referred to as the “trio” of samples:
 Potential father
 Mother
 Child
 In some cases, the mother’s sample was not available
and testing was only done between potential father
and child. This was referred to as “duo” testing.

12. VALIDIT Y OF MOTHERLESS PATERNIT Y
TESTING
 It was concluded that the results from duo, rather
than trio, testing using the AmpFLSTR® Identifiler®
kit were not sufficient to prove paternity:
 In trio cases, the mean value of probability of paternity was
99.999997%, with a minimum of four excluding loci
 In duo cases, over half of the cases were unable to reach the
accepted (99.999%) probability of paternity value.
 This is logical due to the fact that in duo
cases, sometimes as few as 1 excluding loci were
found
 Consider: Do other kits have similar results when
testing in this manner?

13. T HE USE OF P ROM E G A C ORPORAT I ON’S POW E RPLE X ™ 16 A N D
A P P LI E D BI OSYST E MS’ A M P F LSTR® SG M P LUS™ I N T H E
A N ALYSIS OF DE G RA DE D DN A SA M P LE S
The author(s) of this study sought to compare
the Powerplex™ 16 and AmpFlSTR® SGM
Plus™ systems’ ability to analyze degraded
DNA samples
Even though the kits did not have all loci in
common, the nine loci they did have in
common were used as a basis for comparison
24 samples were analyzed

14. ANALYSIS OF DEGRADED DNA SAMPLES AS
MEASURED BY VALID LOCI RESULTS
Loci Powerplex™ 16 (%) AmpFlSTR® SGM Plus™ (%)
D3S1358 21 (88) 20 (83)
vWA 21 (88) 24 (100)
D16S539 21 (88) 23 (96)
D8S1179 20 (83) 24 (100)
D2S11 20 (83) 20 (83)
D18S51 15 (63) <-compare-> 7 (29)
THO1 23 (96) 20 (83)
FGA 14 (58) 11 (46)
Amelogenin 19 (79) <--compare-> 23 (96)
• On average/overall, the two kits were similar (80 versus 81% valid
results)
• Differentiation between kits happened more on the level of
individual loci
• Assumption that a sample size of 24 is “acceptable” to draw
general conclusions

15. ADDITIONAL CONSIDERATION: TEMPERATURE
 As a side note, this study also compared the loss of
information when samples were stored at different
temperatures other than the (assumed) average storage
value:
Storage Temperature Powerplex™ 16 AmpFlSTR® SGM Plus™
Room (20-25°C) 18% 7%
4°C 38% 23%
 This suggests that storage temperature has a greater
impact on the Powerplex™ 16 kit than on AmpFlSTR®
SGM Plus™

16. GENOT YPING INCONSISTENCIES - AMPFLSTR®
IDENTIFILER® VS. POWERPLEX® 16
13 STRs were used to compare since they
were shared between both kits
Because different kits use different primers, it
was believed that certain inconsistencies
might exist between them
This issue can be important due to the
common Forensic practice of “databasing”
Some kits that are produced by the same
company can still have primers that are
designed differently

17. GENOT YPIC DISCORDANCES AND FALSE
HOMOZYGOSITIES BETWEEN STR KITS
STR/alleles AmpFLSTR® Identifiler® Powerplex™ 16 Inconsistencies
D5S818 11-12 12 1
10-12 12 3
10-13 13 1
12 12-13 1
10-11 11 3
9-11 9 1
D8S1179 14 14-16 1
FGA 25-26 26 1
D16S539 9-11 9 2
10-11 10 2
VWA 16 16-18 2
16 16-19 1
16 16-17 1
18 16-18 1
18 17-18 1
• Five of the thirteen shared loci showed genetic discrepancies
• Of the remaining inconsistencies, most were false homozygosities caused by the
AmpFLSTR® Identifiler® at the D5S818 loci
• Alternatively, all identified inconsistencies present at the VWA loci were a result
of the Powerplex™ 16 kit

18. AN OVERVIEW OF FINDINGS
AMPFISTR® SGM Plus™ AmpFlSTR Profiler Plus™ AmpFLSTR® Identifiler® Powerplex™ 16
• Effective at • Most effective at • Not suitable to • Roughly 80%
analyzing LCN analyzing 1-2ng of determine paternity accuracy when
samples if DNA without maternal analyzing
approached correctly • Gains roughly a 15% sample degraded DNA
• Showed better identification rate • Falls within acceptable • Displays less false
results for valid when coupled with paternity findings when homozygosities in
detection of sex- the microcon-30 analyzing the standard the D5S818 loci
determination loci, purification unit “trio” samples than the
Amelogenin • Unable to detect • More relatively AmpFLSTR®
• Roughly 80% most loci in amounts accurate at typing the Identifiler® kit
accuracy when of sample around VWA loci than • More relatively
analyzing degraded 0.015 ng or less Powerplex™ 16 affected by
DNA samples storage
• Retention of temperature of
information is less samples than
sensitive to AmpFLSTR®
temperature in Identifiler® kit
sample storage

19. REFERENCES
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