Ampflstr Identifiler Kit Innovations

Applied Biosystems introduced two new technologies with their AmpFlSTR® Identifiler™ kit when it was released in 2001. The first, and most obvious,

Figure 5.6

AmpFlSTR® Identifier™ allelic ladders (Applied Biosystems). A total of 205 alleles are included in this set of allelic ladders used for genotyping a multiplex PCR reaction involving 15 STR loci and the amelogenin sex-typing test.

Table 5.4 (facing)

Table 5.4

Comparison of represented alleles in commercially available STR allelic ladders

Table 5.4

Comparison of represented alleles in commercially available STR allelic ladders

Promega Corporation STR Kits

Applied Biosystems AmpFlSTR Kits

Loci/Kit

PP1.1 Alleles

#

PP2.1 Alleles

#

PP16 Alleles

#

PP ES Alleles

#

ProfilerPlus Alleles #

COfiler Alleles #

SGM Plus Alleles

#

Identifiler Alleles

#

SEfiler Alleles

#

CSF1PO

6-15

10

6-15

10

6-15 10

6-15

10

FGA

17-46.2

19

16-46.2

28

16-46.2

28

17-30

14

17-51.2

28

17-51.2

28

17-51.2

28

TH01

5-11

7

4-13.3

10

4-13.3

10

4-13.3

10

5-9.3,10 7

4-13.3

10

4-13.3

10

4-13.3

10

TPOX

6-13

8

6-13

8

6-13

8

6-13 8

6-13

8

VWA

10-22

13

10-22

13

10-22

13

10-22

13

11-21

11

11-24

14

11-24

14

11-24

14

D3S1358

12-20

9

12-20

9

12-20

9

12-19

8

12-19 8

12-19

8

12-19

8

12-19

8

D5S818

7-15

9

7-16

10

7-16

10

7-16

10

D7S820

6-14

9

6-14

9

6-15

10

6-15 10

6-15

10

D8S1179

7-18

12

7-18

12

7-18

12

8-19

12

8-19

12

8-19

12

8-19

12

D13S317

7-15

9

7-15

9

8-15

8

8-15

8

D16S539

5,8-15

9

5,8-15

9

5,8-15 9

5,8-15

9

5,8-15

9

5,8-15

9

D18S51

8-27

22

8-27

22

8-27

22

9-26

21

7,9-27

23

7,9-27

23

7,9-27

23

D21S11

24-38

24

24-38

24

24-38

24

24.2-38

22

24-38

24

24-38

24

24-38

24

D2S1338

15-28

14

15-28

14

15-28

14

D19S433

9-17.2

15

9-17.2

15

9-17.2

15

Penta D

2.2-17

14

Penta E

5-24

20

5-24

20

SE33

4.2-37

35

4.2-37

35

Amelogenin

X,Y

2

X,Y

2

X,Y

2

X,Y

2

X,Y 2

X,Y

2

X,Y

2

X,Y

2

Total Alleles

76

137

209

155

118

54

159

205

Illustration of mobility modifiers used in Applied Biosystems ' Identifiler STR kit. Non-nucleotide linkers are synthesized into the primer between the fluorescent dye and 5'-end of the primer sequence. During PCR amplification, the dye and linker are incorporated into the amplicon. With the added non-nucleotide linker, the mobility of the generated STR allele will be shifted to a larger apparent size during electrophoresis. This shift of STR alleles for a particular locus then enables optimal inter-locus spacing for STR loci labeled with the same fluorescent dye without having to alter the PCR primer binding positions (see Figure 5.8).

involves the use of 5-dye detection systems (see Chapter 13) where four different dyes (6FAM™, VIC™, NED™, and PET™) are used to label the PCR products rather than the traditional three dyes (5FAM, JOE, NED or FL, JOE, TMR) as used with the previous AmpFlSTR or PowerPlex kits. A one dye detection channel is always used for an internal size standard to correlate electrophoretic mobilities to an apparent PCR product size (see Chapter 15). Thus, the fifth dye (LIZ™) in 5-dye detection and the fourth dye (ROX or CXR) in 4-dye detection are used for labeling the internal size standard. The extra dye channel for labeling PCR products enables smaller PCR products to be generated and placed in a separate dye channel rather than extending the size range for amplicons within the three previously available dye channels.

The second technology introduced with the Identifiler™ kit involves mobility modifying non-nucleotide linkers (Applied Biosystems 2001). The mobility modifier is composed of hexaethyleneoxide (HEO) that imparts a shift of approximately 2.5 nucleotides with each additional HEO unit (Grossman et al. 1994). This non-nucleotide linker is synthesized into the 5'-end of the PCR primer so that when the PCR product is created it contains these extra molecules on one end (Figure 5.7). By incorporating non-nucleotide linkers, mobilities for amplified alleles from one member of a pair of closely spaced STR loci can be shifted relative to the other. Thus, overlapping size ranges can be prevented (Figure 5.8).

The primary reason for introducing mobility modifiers is to permit continued use of the same PCR primers for amplifying STR loci and still have optimal inter-locus spacing within the various color channels. For example, if the loci D7S820 and CSF1PO, which are labeled with two different fluorophores in the COfiler kit and therefore do not interfere with one another, were labeled with the same colored fluorescent label (e.g., 6FAM) as they are in the Identifiler STR kit, the allelic ladder products would have overlapped by ~13 bp (Figure 5.8). To prevent this overlap in allele size ranges, either PCR primer binding sites must be altered to change the overall size of the PCR product or mobility

Size overlap

(a) COfiler kit allele relative size ranges

D7S820

NED-labeled (yellow)

256.01 bp

Size overlap

(a) COfiler kit allele relative size ranges

D7S820

NED-labeled (yellow)

CSF1PO

labeled (green)

317.67 bp

CSF1PO

labeled (green)

317.67 bp

(b) Identifiler kit allele relative size ranges

10 non-nucleotide linkers

D7S820

CSF1PO

1 6FAM-labeled (blue) 1 1 6FAM-labeled (blue)

255.15 bp 291.58 bp 304.69 bp 341.84 bp

modifiers can be introduced to shift the apparent molecular weight of the larger PCR product to an even larger size. In the case of the Identifiler™ kit, the locus CSF1PO was shifted by approximately 25 bp - most likely through the addition of 10 HEO non-nucleotide linkers to the 5'-end of the labeled PCR primer. Non-nucleotide linkers are also present on four other loci in the Identifiler™ kit: D2S1338, D13S317, D16S539, and TPOX.

Promega has changed primer sequences for a few of the loci between PowerPlex versions (see Masibay et al. 2000, Butler et al. 2001, Krenke et al. 2002). For example, between the PowerPlex® 1.1 and PowerPlex® 16 kits, the CSF1PO primer positions were drastically altered in order to achieve a 30 bp shift in PCR product size between the two kits (Figure 5.9). This primer change and subsequent PCR product shift was instituted so that CSF1PO and D16S539 loci could be labeled with the same dye in the PowerPlex® 16 kit. Note that if the original CSF1PO primers had been kept, there would have been a 13 bp overlap between D16S539 allele 15 (304bp) and CSF1PO allele 6 (291 bp) making these systems incompatible in the same dye color without altering the PCR product size (i.e., primer positions) for one of them.

As will be discussed in Chapter 6, different primer positions have the potential to lead to allele dropout if a primer binding site mutation impacts one of the primer pairs. Hence concordance studies are needed between various STR kits to assess the level of potential allele dropout (Budowle et al. 2001). On the other hand, Applied Biosystems has maintained the same primers over time

Figure 5.8

Illustration of how non-nucleotide linkers attached to CSF1PO PCR products in the Identifiler STR kit help with inter-locus spacing between D7S820 and CSF1PO. In the COfiler kit (a), CSF1PO and D7S820 are labeled with different colored fluorescent labels and thus do not interfere with one another. However, in the Identifiler kit (b), both D7S820 and CSF1PO are labeled with the same dye and would therefore have overlapping STR alleles unless primer positions were changed or mobility modifiers were used. A ~25 bp shift of the CSF1PO PCR products is accomplished by the addition of 10 non-nucleotide linkers. PCR product sizes for allelic ladder ranges displayed here are from the COfiler and Identifiler kit user's manuals. Note that sizes for D7S820 alleles do not match exactly because different dye labels are used with both the PCR products and the internal size standard thus impacting their relative mobilities.

Figure 5.9

Variation in CSF1PO primer positions between (a) PowerPlex 1.1 and (b) PowerPlex 16 STR kits. The base pair (bp) numbers in bold indicate the distance between the repeat region and 3'-end of the pertinent primer. The overall PCR product size for CSF1PO is shifted +30 bp with the primer changes from PowerPlex 1.1 to PowerPlex 16.

CSF1PO

forward primer ZAP ATI

CSF1PO

reverse primer ->

PCR product sizes = 291-327 bp

CSF1PO forward primer

JOE-labeled 238 bP 13 bP

CSF1PO reverse primer

+30 bp shift in size|

and through their various AmpFlSTR® kits (Holt et al. 2002) by introducing

5-dye chemistry and mobility modifiers for products that would normally overlap with one another (see Figure 5.8).

DETAILS ON ALLELES PRESENT IN THE 13 CODIS STR LOCI

Each of the 13 core STR loci has unique characteristics, either in terms of the number of alleles present, the type of repeat sequence, or the kinds of microvariants that have been observed. This section reviews some of the basic details on each of the 13 core STR loci. We have included in Appendix I a detailed summary of the alleles that have been reported as ofJune 2004 for the 13 core STR loci along with their expected sizes using various kits that are available from Promega or Applied Biosystems. The size difference in the PCR products produced by the different STR kits is important because a large difference is more likely to lead to null alleles when comparing results between two kits (see Chapter 6).

CSF1PO is a simple tetranucleotide repeat found in the sixth intron of the c-fms proto-oncogene for the CSF-1 receptor on the long arm of chromosome 5. Common alleles contain an AGAT core repeat and range in size from

6-15 repeats. An allele 16 has also been reported (Margolis-Nunno et al. 2001)

as have several x.1 and x.3 variant alleles (see Appendix I). PCR products from Promega's PowerPlex® 1.1 STR kit are 11 bp larger than those generated with Applied Biosystems kits for equivalent alleles. Since PowerPlex 16 adds 30 bp to the size of CSF1PO relative to PowerPlex® 1.1 (see Figure 5.9), then PowerPlex® 16 PCR products for CSF1PO are 41 bp larger than those generated with AmpFlSTR® kits.

FGA is a compound tetranucleotide repeat found in the third intron of the human alpha fibrinogen locus on the long arm of chromosome 4. FGA has also been referred to in the literature as FIBRA or HUMFIBRA. The locus contains a CTTT repeat flanked on either side by degenerate repeats. The spread in allele sizes is larger for FGA than any of the other core STR loci. Reported alleles range in size from 12.2 repeats to 51.2 repeats, spanning over 35 repeats! A 2 bp deletion, from the loss of a CT, in the region just prior to the core repeat motif is responsible for the x.2 microvariant alleles that are very prevalent in this STR system. PCR products from Promega's PowerPlex® 2.1 and 16 STR kits are 112 bp larger than those generated with Applied Biosystems AmpFlSTR® kits for equivalent alleles. This size difference between these two primer sets is the largest of any of the 13 core loci. So far a total of 80 different FGA alleles have been reported (see Appendix I) making it one of the most polymorphic loci used in human identity testing.

TH01 is a simple tetranucleotide repeat found in intron 1 of the tyrosine hydroxylase gene on the short arm of chromosome 11. The locus name arises from the initials for tyrosine hydroxylase and intron 1 (i.e., 01). The locus is sometimes incorrectly referred to as 'THO1' with an 'O' instead of a 'zero.' In the literature, TH01 has also been referred to as TC11 and HUMTH01.

TH01 has a simple tetranucleotide sequence with a repeat motif of TCAT on the upper strand in the GenBank reference sequence. The repeat motif is commonly referenced as AATG, which is correct for the complementary (bottom) strand to the GenBank reference sequence. A common microvariant allele that exists in Caucasians contains a single base deletion from allele 10 and is designed allele 9.3. Other x.3 alleles have been reported such as 8.3, 10.3, and 13.3 (Griffiths et al. 1998). TH01 has probably been the most studied of the 13 core loci with over 1000 population studies reported in the literature using this DNA marker. PCR products from Promega's PowerPlex® 1.1 STR kit are 11 bp larger than those generated with Applied Biosystems AmpFlSTR® kits for equivalent alleles. PowerPlex® 2.1 STR kits produce amplicons that are 19 bp smaller than PowerPlex® 1.1. The PowerPlex® 2.1 and PowerPlex® 16 STR kits contain the same PCR primers for TH01.

TPOX is a simple tetranucleotide repeat found in intron 10 of the human thyroid peroxidase gene near the very end of the short arm of chromosome 2. TPOX has also been referred to in the literature as hTPO. This STR locus possesses a simple AATG repeat and is the least polymorphic of the 13 core loci. PCR products from Promega's PowerPlex™ 1.1 STR kit are 7 bp larger than those generated with Applied Biosystems AmpFlSTR® kits for equivalent alleles. PowerPlex® 2.1 STR kits produce amplicons that are 38 bp larger in size relative to PowerPlex® 1.1. The PowerPlex® 2.1 and PowerPlex® 16 STR kits contain the same PCR primers for TPOX. Tri-allelic (three banded) patterns are more prevalent in TPOX than any other forensic STR marker (see Chapter 6).

VWA is a compound tetranucleotide repeat found in intron 40 of the von Willebrand Factor gene on the short arm of chromosome 12. VWA has also been referred to in the literature as vWF and vWA. It possesses a TCTA repeat interspersed with a TCTG repeat. The VWA marker targeted by STR multiplex kits is only one of three repeats present in that region of the von Willebrand Factor. The other two have not been found to be as polymorphic (Kimpton et al. 1992). PCR products from Promega's PowerPlex® 1.1 STR kit are 29 bp smaller than those generated with Applied Biosystems AmpFlSTR® kits for equivalent alleles. The PowerPlex® 1.1 and PowerPlex® 2.1 STR kits overlap at three STRs including VWA. Both kits produce amplicons that are equivalent in size for VWA alleles. The PowerPlex® 2.1 and PowerPlex® 16 STR kits contain the same PCR primers for VWA.

D3S1358 is a compound tetranucleotide repeat found on the short arm of chromosome 3. This locus possesses both AGAT and AGAC repeat units (Mornhinweg et al. 1998). The D3 marker is common to Applied Biosystems AmpFlSTR® multiplexes Blue™, Profiler™, Profiler Plus™, COfiler™, SGM Plus™, SEfiler™, and Identifiler™. PCR products from Promega's PowerPlex® 2.1 STR kit are 2 bp larger than those generated with Applied Biosystems kits for equivalent alleles. The PowerPlex® 2.1 and PowerPlex® 16 STR kits contain the same PCR primers for D3S1358.

D5S818 is a simple tetranucleotide repeat found on the long arm of chromosome 5. The locus possesses AGAT repeat units with alleles ranging in size from 7-16 repeats. In both Promega and Applied Biosystems STR kits, D5S818 is one of the smaller sized loci and as such should appear more than some of the other loci in degraded DNA samples. Only a few rare microvariants have been reported at this STR marker. PCR products from Promega's PowerPlex® 1.1 STR kit are 15 bp smaller than those generated with Applied Biosystems kits for equivalent alleles and PowerPlex® 16 retains the original PowerPlex® 1.1 primers.

D7S820 is a simple tetranucleotide repeat found on the long arm of chromosome 7. The locus possesses primarily a GATA repeat. However, a number of new D7 microvariant alleles have been reported recently (see Appendix I). These x.1 and x.3 alleles likely result due to a variation in the number of T nucleotides found in a poly(T) stretch that occurs 13 bases downstream of the core GATA repeat. Sequencing has revealed that 'on-ladder' alleles contain nine tandem T's while x.3 alleles contain eight T's and x.1 alleles contain 10 T's (Egyed et al. 2000). PCR products from Promega's PowerPlex® 1.1 STR kit are 42 bp smaller than those generated with Applied Biosystems kits for equivalent alleles.

D8S1179 is a compound tetranucleotide repeat found on chromosome 8. In early publications by the Forensic Science Service, D8S1179 is listed as D6S502 because of a labeling error in the Cooperative Human Linkage Center database from which this STR was chosen (Oldroyd et al. 1995, Barber and Parkin 1996). The locus consists primarily of alleles containing TCTA although a TCTG repeat unit enters the motif for all alleles larger than 13 repeats, usually at the second or third position from the 5'-end of the repeat region (Barber and Parkin 1996). PCR products from Promega's PowerPlex® 2.1 and PowerPlex® 16 STR kits are 80 bp larger than those generated with Applied Biosystems kits for equivalent alleles. AmpFlSTR® Identifiler™ and Profiler Plus™ ID kits possess an extra, unlabeled D8S1179 reverse primer to prevent allele dropout in Asian populations due to a mutation in the middle of the primer-binding site (Leibelt et al. 2003).

D13S317 is a simple TATC tetranucleotide repeat found on the long arm of chromosome 13. Common alleles contain between 7-15 repeat units although alleles 5, 6, and 16 have been reported (see Appendix I). PCR products from Promega's PowerPlex® 1.1 STR kit are 36 bp smaller than those generated with Applied Biosystems AmpFlSTR® kits for equivalent alleles. A 4 bp deletion has been reported 24 bases downstream from the core TATC repeat that can impact allele calls with different primer sets (Butler et al. 2003, Drabek et al. 2004). PowerPlex® 16 primers, while generating the same size amplicons as the original PowerPlex® 1.1 primers, have been shifted to avoid this 4 bp deletion that is present in some African-American samples.

D16S539 is a simple tetranucleotide repeat found on the long arm of chromosome 16. Nine common alleles exist that possess a core repeat unit of GATA. These include an allele with five repeats and consecutive alleles ranging from 8-15 repeat units in length. PCR products from Promega STR kits are 31 bp larger than those generated with Applied Biosystems kits for equivalent alleles. A point mutation (T^A) 38 bp downstream of the STR repeat impacts the reverse primers for both Applied Biosystems and Promega primer sets. Applied Biosystems added an extra or 'degenerate' unlabeled primer in their COfiler™, SGM Plus™, and Identifiler™ kits so that both possible alleles could be amplified (Wallin et al. 2002). On the other hand, Promega altered their D16S539 reverse primer sequence between kits but kept the overall amplicon size the same (Butler et al. 2001). The 3'-end of the PowerPlex® 1.1 reverse primer was lengthened by five nucleotides to create the PowerPlex® 16 reverse primer and thus move the primer mismatch caused by this mutation further into the primer to prevent allele dropout (Nelson et al. 2001, Krenke et al. 2002).

D18S51 is a simple tetranucleotide repeat found on the long arm of chromosome 18. It has a repeat motif of AGAA. A number of x.2 allele variants exist due to a 2 bp deletion from a loss of AG in the 3'-flanking region (Barber and Parkin 1996). More than 50 alleles have been reported for D18S51 making it one of the more polymorphic of the 13 core loci. PCR products from Promega's PowerPlex® 2.1 STR kit are 22 bp larger than those generated with Applied Biosystems AmpFlSTR® kits for equivalent alleles. The PowerPlex® 2.1 and PowerPlex® 16 STR kits contain the same PCR primers for D18S51.

D21S11 is a complex tetranucleotide repeat found on the long arm of chromosome 21. A variable number of TCTA and TCTG repeat blocks surround a constant 43 bp section made up of the sequence {[TCTA]3 TA [TCTA]3 TCA [TCTA]2 TCCA TA}. The x.2 microvariant alleles arise primarily from a 2 bp (TA) insertion on the 3'-end of the repeat region (Brinkmann et al. 1996). PCR products from Promega's PowerPlex® 2.1 STR kit are 17 bp larger than those generated with Applied Biosystems AmpFlSTR® kits for equivalent alleles. The PowerPlex® 2.1 and PowerPlex® 16 STR kits contain the same PCR primers for D21S11.

Early papers in the literature by the Forensic Science Service had alleles named based on the dinucleotide subunit CV, where the V represents either an A, T, or G (Urquhart et al. 1994), while other authors adopted a different allele naming scheme based on the primary tetranucleotide repeat (Moller et al. 1994). As outlined in the European DNA Profiling Group inter-laboratory study on D21S11 (Gill et al. 1997), a simple formula can be used to convert the Urquhart (U) designation into the Moller (M) equivalent:

Today most laboratories use the Moller allele notation since it fits the ISFG allele designation recommendation (Bar et al. 1997).

D21S11 is far more polymorphic than can be easily detected with sized-based length separations. A careful search of the literature has revealed more than 80 reported alleles, many of which are the same length (see Appendix I). Fine differences in the D21S11 allele structures can only be determined by DNA sequencing since so many of the alleles have the same length but different internal sequence structure because some of the repeat units are switched around. For example, there are four different alleles designated as 30 repeats, which are indistinguishable by size-based methods alone (Appendix I).

The three most polymorphic of the 13 loci are D21S11, FGA, and D18S51. These loci contain numerous microvariant alleles that are being uncovered as more and more samples are examined around the world.

ADDITIONAL STR LOCI COMMONLY USED

The 13 core loci used within the United States for CODIS are effective DNA markers for human identification and will most likely continue to be used for some time. However, these 13 markers are by no means the only STRs that have been evaluated or used by forensic labs around the world. Dozens of other markers have been used, some quite extensively (Table 5.5).

Table 5.5 (below) Some additional STR markers used in the forensic DNA community. STR markers in bold are part of commonly used multiplex kits. For information on Y chromosome STRs, see Tables 9.2 and 9.5.

Locus

Chromosomal

GenBank

Repeat

Allele

Amplicon

Reference

Name

Location

Accession

ISFG format

Range

Size Range

ARA

Xcen-q13

M21748

CAG

14-32

255-315 bp

Hammond et al. (1994)

APOAI1

11q23-qter

J00048

AAAG

Complex

263-291 bp

Dupuy and Olaisen (1997)

ACTBP2

6

V00481

AAAG

4.2-37

198-325 bp

Dupuy and Olaisen (1997)

CD4

12p12-pter

M86525

TTTTC

6-16

125-175 bp

Hammond et al. (1994)

CYAR04

15q21.1

M30798

AAAT

5-12

173-201 bp

Hammond et al. (1994)

F13A01

6p24.3-25.1

M21986

GAAA

3.2-16

281-331 bp

Hammond et al. (1994)

F13B

1q31-q32.1

M64554

TTTA

6-12

169-193 bp

Promega

FABP

4q28-31

M18079

ATT

10-15

199-220 bp

Hammond et al. (1994)

FES/FPS

15q25-qter

X06292

ATTT

7-14

222-250 bp

Hammond et al. (1994)

HPRTB

Xq26.1

M26434

TCTA

6-17

259-303 bp

Hammond et al. (1994)

LPL

8p22

D83550

TTTA

7-14

105-133 bp

Promega

Penta D

21q

AP001752

AAAGA

2.2-17

376-449 bp

PowerPlex 16

Penta E

15q

AC027004

AAAGA

5-24

379-474 bp

PowerPlex 16

PLA2A1

12q23-qter

M22970

AAT

118-139 bp

Hammond et al. (1994)

RENA4

1q32

M10151

ACAG

255-275 bp

Hammond et al. (1994)

D1S1656

1 pter-qter

G07820

(TAGA) (TAGG)

9-19.3

125-168 bp

Wiegand et al. (1999)

D2S1242

2pter-qter

L17825

(GAAA) (GAAG)

1 0-1 8

141-175 bp

Reichenpfader et al. (1999)

D2S1338

2q35-37.1

G08202

(TGCC) (TTCC)

15-28

289-341 bp

SGM Plus, Identifiler

D3S1359

3p

AA306290

TCTA

11-25.3

196-255 bp

Poltl et al. (1998)

D3S1744

3q24

G08246

GATA

14-22

150-182 bp

Lifecodes

Locus Name

Chromosomal Location

GenBank Accession

Repeat ISFG format

Allele Range

Amplicon Size Range

Reference

D6S477

6pter-qter

G08543

TCTA

13.2-22

206-240 bp

Carracedo and Lareu (1998)

D7S809

7pter-qter

X73290

(AGGA) (AGGC)

9 alleles

241-289 bp

Tamaki et al. (1996)

D8S347

8q22.3-24.3

L12268

AGAT

16-28

340-388 bp

Poltl et al. (1997)

D8S639

8p21-p11

L24797

(AGAT) (AGGT)

20-33.3

316-371 bp

Seidl et al. (1999)

D9S302

9q31-33

G08746

ATCT

17 alleles

255-353 bp

Carracedo and Lareu (1998)

D10S2325

10pter-qter

G08790

TCTTA

6-17

113-168 bp

Wiegand et al. (1999)

D11S488

11 q24.1-25

L04732

(AAAG) (GAAG)

26-41

242-302 bp

Seidl et al. (1999)

D11S554

11p11.2-12

M87277

AAAG

Complex

176-286 bp

Dupuy and Olaisen (1997)

D12S391

12

G08921

(AGAT) (AGAC)

15-26

209-253 bp

Lareu et al. (1996)

D12S1090

12q12

Not found

GATA

9-33

212-306 bp

Lifecodes

D18S535

18pter-qter

G07985

GATA

9-16

130-158 bp

Wiegand et al. (1999)

D18S849

18q12-q21

G07992

GATA

9-20

93-133 bp

Lifecodes

D19S433

19q12-13.1

G08036

AAGG

9-17.2

106-140 bp

SGM Plus, Identifiler

D20S161

20pter-qter

L16405

TAGA

14-22

156-187 bp

Hou et al. (1999)

D22S683

22pter-qter

G08086

(TA) (TATC)

12-21.2

168-206 bp

Carracedo and Lareu (1998)

DXS6807

Xpter-p22.2

G09662

GATA

11-17

251-275 bp

Edelmann and Szibor (1999)

Table 5.5 Applied Biosystems has created the AmpFlSTR® SGM Plus™ kit that co-amplifies

(cContinued)) 10 STR loci including two new STRs: D19S433 and D2S1338. With the adoption of the SGM Plus kit by the Forensic Science Service and much of Europe, the amount of population data on the STR loci D19S433 and D2S1338 will continue to grow. These two loci are also part of the Identifiler 16plex STR kit. Likewise, the Promega Corporation has included two pentanucleotide STR loci, Penta E and Penta D, in their GenePrint® PowerPlex® 2.1 and PowerPlex® 16 kits. Because these markers are included in the STR multiplexes in conjunction with the 13 core loci for developing DNA databases, they will become more prevalent as the number of samples in the databases grows.

Owing to the fact that the German national DNA database requires analysis of the complex hypervariable STR locus SE33, Promega created the PowerPlex®

Table 5.5 Applied Biosystems has created the AmpFlSTR® SGM Plus™ kit that co-amplifies

(cContinued)) 10 STR loci including two new STRs: D19S433 and D2S1338. With the adoption of the SGM Plus kit by the Forensic Science Service and much of Europe, the amount of population data on the STR loci D19S433 and D2S1338 will continue to grow. These two loci are also part of the Identifiler 16plex STR kit. Likewise, the Promega Corporation has included two pentanucleotide STR loci, Penta E and Penta D, in their GenePrint® PowerPlex® 2.1 and PowerPlex® 16 kits. Because these markers are included in the STR multiplexes in conjunction with the 13 core loci for developing DNA databases, they will become more prevalent as the number of samples in the databases grows.

Owing to the fact that the German national DNA database requires analysis of the complex hypervariable STR locus SE33, Promega created the PowerPlex®

Figure 5.10

SE33 (ACTBP2) allelic ladder from PowerPlex ES kit produced by the Promega Corporation.

ES kit in 2001 and Applied Biosystems released the SEfiler™ kit in late 2002 to provide SE33 in a commercial kit form. The PowerPlex® ES allelic ladder for SE33 contains 35 alleles, which demonstrates this STR marker's variability (Figure 5.10).

Promega also has a multiplex commonly referred to as FFFL, which is used by many laboratories in South America to amplify the four STRs F13A01, F13B, FES/FPS, and LPL. Table 5.5 includes a listing of these markers as well as many others that have appeared in the literature along with useful information such as the GenBank accession number, references, and size ranges with a reported set of PCR primers. STR markers on the Y chromosome are described in Chapter 9. These Y-STRs are becoming increasingly popular due to their ability to aid sexual assault investigations through male-specific amplification.

Figure 5.10

SE33 (ACTBP2) allelic ladder from PowerPlex ES kit produced by the Promega Corporation.

GENDER IDENTIFICATION WITH AMELOGENIN

The ability to designate whether a sample originated from a male or a female source is useful in sexual assault cases, where distinguishing between the victim and the perpetrator's evidence is important. Likewise, missing persons and mass disaster investigations can benefit from gender identification of the remains. Over the years a number of gender identification assays have been demonstrated using PCR methods (Sullivan et al. 1993, Eng et al. 1994, Reynolds and Varlaro 1996). By far the most popular method for sex-typing today is the amelogenin system as it can be performed in conjunction with STR analysis.

Amelogenin is a gene that codes for proteins found in tooth enamel. The British Forensic Science Service was the first to describe the particular PCR primer sets that are used so prevalently in forensic DNA laboratories today (Sullivan et al. 1993). These primers flank a 6 bp deletion within intron 1 of the amelogenin gene on the X homologue (Figure 5.11). PCR amplification of this area with their primers results in 106 bp and 112 bp amplicons from the X and

Figure 5.11 Schematic of the amelogenin sex-typing assay. The X and Y chromosomes contain a high degree of sequence homology at the amelogenin locus. The primer sets depicted here target a 6 bp deletion that is present only on the X chromosome. The presence of a single peak indicates that the sample comes from a female while two peaks identifies the sample's source as male. The primers to amplify the 106/112bp fragments are used in the AmpFlSTR kits while the PowerPlex 1.1 kit uses the larger primer set.

AmpFISTR kits and PowerPlex 16

PowerPlex 1.1

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