Identification of a D8S1179 primer binding site mutation and the validation of a primer designed to recover null alleles

Forensic Science International 133 (2003) 220–227

Identification of a D8S1179 primer binding site mutation and the validation of a primer designed to recover null alleles Craig Leibelta,*, Bruce Budowleb, Patrick Collinsa, Yasser Daoudia, Tamyra Morettic, Gary Nunna, Dennis Reedera, Rhonda Robya a

Applied Biosystems, 850 Lincoln Centre Drive, M/S 404-3, Foster City, CA 94404, USA b Laboratory Division, FBI, 935 Pennsylvania Ave., NW, Washington, DC 20535, USA c Laboratory Division, FBI Academy, Quantico, VA 22135, USA

Received 25 October 2002; received in revised form 27 January 2003; accepted 29 January 2003

Abstract A population study of Chamorros and Filipinos using short tandem repeat (STR) loci amplified with the AmpF‘STR1 Profiler PlusTM PCR amplification kit demonstrated an excess of observed homozygosity at the D8S1179 locus. Use of a different set of D8S1179 primers to type the same samples did not demonstrate an excess of homozygosity and showed discordant genotypes at the D8S1179 locus. A single point mutation, G-to-A transition, 16 nucleotides from the 30 end of the reverse primer, was identified to cause allele dropout when using the AmpF‘STR1 Profiler PlusTM primer set. An additional D8S1179 reverse primer specific for the variant was constructed resulting in the recovery of the null allele. The primer was included in the newly developed AmpF‘STR1 IdentifilerTM PCR amplification kit. No deleterious effects or non-specific peaks were observed in validation experiments evaluating primer concentration, Mg2þ concentration, annealing temperature and population samples. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: D8S1179; Primer binding site mutation; AmpF‘STR1; Multiplex; PCR; STR; Validation

1. Introduction The PCR amplification and detection of fluorescent dye labeled short tandem repeat (STR) loci are now commonplace in identifying human remains and forensic specimens. The combined DNA index system (CODIS) requires that input of offender samples for comparison into the national database contain, at minimum, 13 specific loci [1] which includes the locus D8S1179 [2]. Commercial kits are available that enable multiplex amplification of the 13 core STR loci required by CODIS [3]. A concordance study comparing genotypes of common loci in two commercial multiplex kits was conducted [4]. The PowerPlex1 16 system (Promega Corp., Madison, WI) was shown to yield discordant D8S1179 profiles on a subset * Corresponding author. Tel.: þ1-650-554-2408; fax: þ1-650-638-6393. E-mail address: [email protected] (C. Leibelt).

of Chamorro and Filipino population samples (N ¼ 140) obtained from the island of Guam when compared with results from the AmpF‘STR1 Profiler PlusTM PCR amplification kit (Applied Biosystems). Thirteen of the observed single peak patterns detected using the AmpF‘STR1 Profiler PlusTM primer set were typed as heterozygotes using the PowerPlex1 16 primers. Previously, Budowle et al. [5] detected a significant departure from Hardy–Weinberg expectations at the D8S1179 locus in the same Chamorro and a Filipino sample populations (P ¼ 0:005, Chamorros; P ¼ 0:030, Filipinos). The observed and expected homozygosity were 38.4 and 22.8%, respectively, for Chamorros and 25.8 and 15.8%, respectively, for Filipinos. This paper describes: (1) the identification of a variant in the primer binding site of the AmpF‘STR1 D8S1179 reverse primer (primer R1) that causes allele dropout; (2) the use of an additional unlabeled D8S1179 reverse primer specific for the variant (primer R2) to recover the loss of the allele; and (3) validation experiments to determine if the addition of

0379-0738/03/$ – see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0379-0738(03)00035-5

C. Leibelt et al. / Forensic Science International 133 (2003) 220–227

221

primer R2 causes any deleterious effect on the performance of the newly developed AmpF‘STR1 IdentifilerTM PCR amplification kit which amplifies 15 STR loci (D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, TH01, TPOX, vWA) and the Amelogenin gender determining marker.

Additionally, three control samples exhibiting no allele dropout at the D8S1179 locus were tested. These included two genomic DNA Caucasian samples and cell line 9947A DNA (AmpF‘STR1 kit control). All samples were extracted using a phenol/chloroform protocol and the quantity of DNA was determined using the QuantiBlot1 Human DNA Quantitation kit (Applied Biosystems) [6].

2. Materials and methods

2.2. Sequencing

2.1. DNA samples

PCR primers designed to flank the D8S1179 primer binding region utilized in the AmpF‘STR1 PCR amplification kits were constructed (Fig. 1). Generation of sequencing template involved two rounds of PCR with band isolation prior to the second-round as previously described by Lazaruk et al. [7]. Extracted genomic DNA (0.25 ng) was amplified in a 50 ml reaction using 20 pmol of each flanking primer in a GeneAmp1 PCR System 9700 (Applied Biosystems). Cycling parameters consisted of a 10 min enzyme activation at 95 8C followed by 40 cycles of: denaturation at 95 8C (30 s); annealing at 55 8C (30 s); extension at 72 8C (1 min); and final extension 72 8C (10 min).

A total of 19 extracted genomic DNA samples exhibiting allele dropout at the D8S1179 locus when amplified with the AmpF‘STR1 Profiler PlusTM kit were used in this study. These samples included: eight Chamorrans and nine Filipinos obtained from the Federal Bureau of Investigation (FBI); and two samples of unknown population origin obtained from the California Department of Justice (DOJ) DNA Laboratory in Berkeley. DNA from 60 population samples obtained from a variety of forensic laboratories was also included in this study as part of the validation experiments.

Fig. 1. Genbank sequence of D8S1179 (accession no. AF216671) showing the sequencing primers (D8seq—forward and D8seq—reverse) and the original AmpF‘STR1 reverse primer (primer R1). The G-to-A transition (IUB code R) at position #143620 located 16 nucleotides from the 30 end of reverse primer R1 is shown boxed. Highlighted only is a D8S1179 allele consisting of a tetranucleotide repeat (tcta)11. Primer R2 designed to amplify the variant and included in the AmpF‘STR1 IdentifilerTM PCR amplification kit is shown at the bottom of the figure.  Primer binding sequence.

222

C. Leibelt et al. / Forensic Science International 133 (2003) 220–227

Two microliters of first-round PCR product were then loaded onto a polyacrylamide gel for subsequent silver staining and band isolation. A 5 ml aliquot of the recovered PCR product was re-amplified (second-round PCR) using the same primers and parameters used for the first-round PCR, except the cycle number was decreased from 40 to 32 cycles. Prior to sequencing, the PCR product was purified using a Microcon1 YM-100 (Millipore Corporation, Bedford, MA). Sequencing was performed using the flanking primers and BigDyeTM Terminator Cycle Sequencing Ready Reaction kits with AmpliTaq1 DNA polymerase FS (Applied Biosystems). Sequence reactions were purified using Millipore MultiScreen1 Assay System (Millipore) loaded with Sephadex1 G-50 Superfine (Amersham Pharmacia Biotech, Uppsala, Sweden). Samples were dried, resuspended in 15 ml of Hi-DiTM Formamide (Applied Biosystems), then subsequently heat denatured at 95 8C for 2 min and snap cooled on ice for 3 min. Electrophoresis and detection were performed on an ABI PRISM1 3100 Genetic Analyzer using data collection v1.0.1 and run module (StdSeq50_POP6DefaultModule). Sequences were analyzed using ABI PRISM1 DNA Sequencing Analysis Software v3.7 (Applied Biosystems). 2.3. Validation experiments The validation experiments presented in this paper relate to a primer designed to recover null alleles. For a discussion of other experiments, see the ‘AmpF‘STR1 IdentifilerTM Users’s Manual’ [8] and Collins et al. [9]. 2.3.1. Amplification Genomic template DNA (0.5–1.0 ng) was amplified in duplicate 25 ml PCRs using reagents supplied in the AmpF‘STR1 IdentifilerTM PCR amplification kit including AmpF‘STR1 PCR reaction mix, AmpF‘STR1 IdentifilerTM primer set (includes dye labeled and unlabeled primers), and AmpliTaq Gold1 DNA polymerase. Two developmental lots of the AmpF‘STR1 IdentifilerTM PCR amplification kit were used in this study: one containing the original AmpF‘STR1 D8S1179 primer set, reverse primer R1 and the forward primer; and one containing the original primer set with the addition of a newly designed unlabeled D8S1179 reverse primer specific for the variant position (primer R2, Fig. 1). The amplification protocol followed the manufacturer’s recommendations (AmpF‘STR1 IdentifilerTM PCR Amplification Kit User’s Manual) [8] using a GeneAmp1 PCR System 9700 (Applied Biosystems). 2.3.2. Capillary electrophoresis and detection All samples were prepared for electrophoresis and loaded onto an ABI PRISM1 310 Genetic Analyzer following the manufacturer’s recommendations (AmpF‘STR1 IdentifilerTM Amplification PCR Kit User’s Manual). All samples were injected twice. Detection was carried out using Data

Collection Software v1.0.1 with module (GS STR Pop 4 (1 ml) G5) and analyzed using Genescan Software v3.1. 2.3.3. Primer input Six samples, four containing the primer binding site mutation and two controls containing the reference primer binding sequence, were amplified using the IdentifilerTM kit with the original D8S1179 primer set. Varying amounts of primer R2 were added to the primer set at the following quantities: 0, 0.4, 1.6, 3.6, 4.5, 6.5, 9.0 (equimolar to primer R1), 13.5 and 18 pmol. Additionally, two reference sequence control samples were amplified in a singleplex reaction using the AmpF‘STR1 kit D8S1179 forward primer and primer R2 only to determine if amplification occurs. 2.3.4. Magnesium chloride input Five samples, two containing the variant and three with the reference sequence, were amplified using the AmpF‘STR1 IdentifilerTM primer set inclusive of primer R2. The MgCl2 was varied in the reaction mix at the following concentrations: 0.5, 1.0, 1.15, 1.25 (standard amount in AmpF‘STR1 PCR reaction mix), 1.35, 1.5, 2.0 and 3.0 mM. 2.3.5. Annealing temperature Four samples, two containing the variant and two with the reference sequence, were amplified using the IdentifilerTM kit with primer R2. Two samples, one with the variant and one with the reference sequence, were amplified using the IdentifilerTM kit without primer R2. Annealing temperatures were varied using the same thermocycler to the following: 55, 57, 58, 59 (standard using AmpF‘STR1 PCR amplification kits), 61 and 63 8C. 2.3.6. Population samples A total of 60 population samples was used to evaluate kit performance and verify genotype concordance. Of the 60 total samples, 20 each were chosen from African-American, Caucasian, and Hispanic populations. From each of these three populations, 10 samples were previously typed as homozygous for the D8S1179 locus. The remaining 10 samples per population were previously typed as heterozygous for the D8S1179 locus. The samples were amplified using the IdentifilerTM kit with and without primer R2. 2.3.7. Null allele samples The 19 samples previously shown to exhibit the D8S1179 primer site variant in the sequencing experiment were amplified using the IdentifilerTM kit with and without primer R2.

3. Results and discussion 3.1. Sequencing All samples available to this analysis that exhibited allele dropout at the D8S1179 locus in the primer concordance

C. Leibelt et al. / Forensic Science International 133 (2003) 220–227

study [4] exhibited a G-to-A transition at position #143620 of the Genbank sequence accession no. AF216671 (Fig. 1). Variants in the primer binding site that affect amplification of an allele have been reported and are to be expected [7,10–18]. A mismatch in the primer binding site can cause: (1) little or no effect; (2) unbalanced heterozygous profiles (lower allele/higher allele  100% < 70%); or (3) the non-amplification of an allele also known as ‘‘allele dropout’’ or ‘‘null allele’’. The outcome depends on amplification conditions, primer length and Tm, and the nature and context of the mismatch. Previous studies have described significant peak height imbalance of heterozygous alleles at STR loci resulting from variants in the primer binding site located three [14] and four [7] nucleotides from the 30 end of a primer. Typically, one would expect variants that cause dropout to reside at or near the 30 end of the primer such as those described as being located at the second [7,11,15] nucleotide from the 30 end of a primer. However, the substitution causing allele dropout at the D8S1179 locus is closer to the 50 end of the reverse primer such as the variant described by Gusmao et al. [16]. The mutation is located 16 nucleotides from the 30 end of the reverse primer. Whereas other authors have reported the occurrence of this D8S1179 variant causing allele dropout [17,18], this is the first report known to the authors where a variant that causes allele dropout of an STR allele has been described as being this distant from the 30 end of a primer. For a primer binding site mutation, particularly distant from the 30 end, to destabilize primer annealing resulting in no extension, the annealing behavior of the primer and the primer binding site must be substantially affected. Three methods including: (1) basic; (2) salt adjusted; and (3) nearest neighbor (two formulas) were used to calculate melting temperatures of primers R1 and R2 to show the effects of the G-to-A transition (Table 1) with no mismatches present. Depending on the method employed, the predicted relative difference in Tm due to the transition can be as much as 2–4 8C. Moreover, the effect on primer annealing resulting from the AC single base pair mismatch can be seen in Table 1 Melting temperatures calculated using a D8S1179 primera concentration of 360 nM and salt concentration of 50 mM (primer R1a vs. primer R2a) Method

Primer R1 Tm (8C)

Primer R2 Tm (8C)

DTm (8C)

Basicb Salt adjustedb Nearest neighborb Nearest neighborc

51 58 56 61.5

49 56 53 57.1

2 2 3 4.4

a

Primer binding sequence. Oligonucleotide Properties Calculator v3.01 last modified 19 December 2000 (http://www.basic.nwu.edu/biotools/oligocalc.html). c Primer Express Software v1.0 (Applied Biosystems). b

223

the annealing temperature experiment. The null allele was restored using the AmpF‘STR1 IdentifilerTM primer set without primer R2 by lowering the annealing temperature from 59 (standard) to 55 8C, although not completely (Fig. 2). A null allele due to a one base pair mismatch can be expected if the Tm has been substantially lowered below the designed annealing temperature (59 8C) of the AmpF‘STR1 kits. The variant described is associated with allele 16 (i.e. containing 16 repeat units) in seventeen out of the 19 sequenced individuals. A single seventeen and an eighteen STR repeat allele also exhibits this mutation. One homozygote sixteen repeat individual is heterozygous for the primer binding site (one allele contained the reference sequence and one allele contained the variant sequence). 3.2. Primer input To overcome the D8S1179 allele dropout, a primer was designed to anneal to the target sequence containing the transition (Fig. 1). Thus, upon the addition of the degenerate primer (primers R1 and R2) to a PCR mixture, the reference sequence allele containing a G and the variant allele containing an A co-amplify if present in the template DNA. Adding a second primer resulting in biallelic degeneracy to a validated multiplex system would not be expected to affect performance and is considered a minor modification according to ‘‘quality assurance standards for forensic DNA testing laboratories’’ [19]. However, validation experiments were carried out for demonstration purposes and quality assurance purposes. These experiments were performed to ensure that no interaction occurs with primer R2 and any of the other primers in the AmpF‘STR1 IdentifilerTM primer set. Also, experiments were performed to show that the addition of primer R2 would have no deleterious effects on, or change the performance of, the current multiplex system. The addition of primer R2 to the AmpF‘STR1 IdentifiTM ler primer set fully recovered the null allele and showed no interaction or deleterious effects such as primer dimer and non-specific amplification. One might expect this finding, since the addition of a primer that differs by a single base substitution from an existing primer in the primer set is a minor change. An increase in the peak heights at the D8S1179 locus was observed in the samples containing the reference sequence. This increase is attributed to primer R2 being capable of annealing to both the reference and variant primer binding site under standard conditions. It would appear that the GT single base pair mismatch is stronger than the AC mismatch in the context tested. PCR amplifications using the original D8S1179 AmpF‘STR1 primer pair does not amplify the variant primer binding site allele while significant amplicon is generated from samples containing the reference sequence using primer R2 and the original forward primer. A similar trend in mismatch stability has been previously reported by Peyret et al. [20], and Hatim and SantaLucia [21,22]. Balanced heterozygote

224

C. Leibelt et al. / Forensic Science International 133 (2003) 220–227

Fig. 2. Electropherograms of amplified DNA containing the primer binding site mutation without primer R2 added to the IdentifilerTM kit. Blue (6-FAMTM dye) labeled loci shown left to right are D8S1179, D21S11 (heterozygote), D7S820 (homozygote) and CSF1PO (heterozygote). The top panel shows an amplification with a standard annealing temperature (TA) of 59 8C. The profile shows the loss of a D8S1179 allele leaving behind a homozygote peak with a significantly lower peak height relative to neighboring loci in same color. The bottom profile shows an amplification with a lowered annealing temperature of 55 8C recovering the null allele.

profiles (peak height ratios >70%) were obtained at 4.5 pmol (i.e. one half the designed primer quantity of 9 pmol) and no deleterious effect was observed as high as 18 pmol thus assuring a window of optimal performance.

in the reaction mix remains unchanged with the addition of primer R2.

3.3. Magnesium chloride input

Altering the annealing temperature affects the stringency of the PCR. The samples herein were amplified over a range of annealing temperatures. The annealing temperature was reduced from 59 (optimal) to 55 8C to test for mispriming resulting from primer R2. In addition, the annealing temperature was increased to 63 8C to assess loss of product. No non-specific peaks were observed at 55 8C. Reduced signal at 61 8C and loss of product at 63 8C was observed using 0.5 ng of genomic template DNA. Minor differences in overall peak heights were observed between IdentifilerTM kits with and without primer R2. Regardless of the two lots tested, the addition of primer R2 appears to have little effect on performance. The optimal annealing temperature of 59 8C remains unchanged with respect to the addition of primer R2.

Increasing Mg2þ decreases the stringency of the PCR [23]; thus primers might anneal to other sites on the template DNA and generate non-specific amplicons. The optimal concentration in the PCR is 1.25 mM [14]. No non-specific peaks were observed up to 3.0 mM. However, allele dropout at the D19S433 locus and a peak at approximately 91 bp that could be attributed to primer dimer was observed at 3.0 mM. This effect is seen in amplifications without the use of primer R2 and is not unexpected due to the low stringency involved. The D8S1179 locus was one of the more robust loci in the multiplex system at the reduced amount of 1.0 mM. The performance window with respect to the optimal concentration of 1.25 mM MgCl2

3.4. Annealing temperature

C. Leibelt et al. / Forensic Science International 133 (2003) 220–227 Table 2 D8S1179 heterozygous peak height ratios using population samples with and without primer R2

N Mean S.D. Range

With primer R2 (%)

Without primer R2 (%)

30 90.49 7.85 62.5–99.57

30 89.26 8.47 68.58–100

3.5. Population samples The genotypes of the 60 population samples were the same in both iterations of the kit. No artifacts or baseline effects were observed due to the addition of primer R2. In addition, no substantial difference was observed in the heterozygous peak height ratios (Table 2). 3.6. Null allele samples In all samples that were previously sequenced and determined to carry the primer binding site mutation, the null alleles were recovered upon using the AmpF‘STR1 IdentifilerTM PCR amplification kit containing primer R2 (Fig. 3).

225

The sample containing a reference and variant 16 allele exhibited a substantial increase in peak height consistent with a true homozygote upon amplification with the IdentifilerTM kit containing the biallelic degenerate primer mixture (Fig. 4). 3.7. Forensic considerations Allele dropout has little effect on a forensic analysis, even at the frequency observed in the Chamorros and Filipinos at the D8S1179 locus. In processing casework, samples typically are amplified using the same kit or primer set. Thus, any primer mismatch present in the reference sample would also be present in an evidence sample that originated from the same individual. Only under rare circumstances would a PCR inhibitor be extracted with evidence DNA resulting in one or more alleles not being exhibited when compared to its reference sample. Discordant genotype results between different primer sets from different manufacturers’ kits will occur at low frequency. This can result in the presence of a pseudo-homozygous sample in a criminal DNA database where a suspect and the evidence sample are typed using two different primers resulting in two different genotypes. However, a mismatch at one locus, particularly if one allele is in common, of a 13 locus

Fig. 3. Electropherograms, blue dye labeled loci only, of amplified genomic DNA containing the primer binding site mutation. The top panel shows an IdentifilerTM kit amplification without primer R2 and the loss a D8S1179 allele. The bottom panel shows an IdentifilerTM kit amplification with primer R2 and a recovery of the null allele.

226

C. Leibelt et al. / Forensic Science International 133 (2003) 220–227

Fig. 4. Electropherograms showing a single D8S1179 peak of an individual containing both the variant and reference primer binding site. The bottom profile (amplification without primer R2) shows a D8S1179 peak with a significantly lower and unbalanced peak height (homozygote similar to heterozygote) relative to neighboring loci in same color. The top panel (amplification with R2) shows an increase in peak height for the D8S1179 peak with a more representative profile compared to the neighboring heterozygous peaks.

profile should still be considered a strong investigative lead. As always, one should review the entire multilocus profile and case circumstances prior to rendering a conclusion. Allele dropout may be misinterpreted as a departure from Hardy–Weinberg expectations and thus one may falsely suggest that the sample population demonstrates substantial substructure. Such an interpretation would be erroneous. Thus, minimizing the degree of allele dropout is useful in preventing such a mistake. However, the effect of allele dropout on estimating the rarity of a multiple locus profile would be nominal. For a 13 locus profile, the average random match probability usually is on the order of 1 in 1012–1015 in most populations [24]. Therefore in previous cases where allele dropout may have occurred, the overall estimate would not be significantly altered. Since the AmpF‘STR1 IdentifilerTM PCR amplification kit contains both reverse primers (primers R1 and R2), the null allele issue for this particular variant is moot. The identification of the AmpF‘STR1 D8S1179 primer binding site mutation and the subsequent recovery of the null allele using the IdentifilerTM kit may be useful in paternity

cases that were previously typed without primer R2. The non-amplification of an allele could produce a type I error (false exclusion). Caution must be used in the interpretation of a single locus mismatch because it is known that mutations and allelic dropout can occur. A false exclusion due to this variant in the D8S1179 locus has now been addressed. To date only the Chamorro and Filipino populations have shown any notable frequency of allele dropout at the D8S1179 locus. The null allele variant has also been observed in a Korean population [17], a Mozambique population [18], and a Japanese population by Toshimichi Yamamoto, Ph.D. from the Nagoya University (personal communication), but at low frequency. When estimating the random match probability for such population groups (or closely related groups) and allele dropout is suspected at the D8S1179 locus, instead of using p þ pð1  pÞy to estimate the genotype frequency, one could use 2pipn where pn is the estimated null allele. Again, this point is not relevant to the use of the AmpF‘STR1 IdentifilerTM PCR amplification kit.

C. Leibelt et al. / Forensic Science International 133 (2003) 220–227

Acknowledgements The authors would like to thank the California Department of Justice DNA Laboratory (Berkeley, CA) for contributing DNA samples exhibiting the mutation and the Serological Research Institute (Richmond, CA) for providing control samples containing the reference sequence. We also wish to thank the Maryland State Police Crime Laboratory (Pikesville, MD) and the Ohio Bureau of Criminal Identification and Investigation (London, OH) for providing the population samples.

[10] [11]

[12] [13]

[14]

References [1] B. Budowle, T.R. Moretti, S.J. Niezgoda, B.L. Brown, CODIS and PCR-based short tandem repeat loci: law enforcement tools, in: Proceedings of the Second European Symposium on Human Identification, Promega Corporation, Madison, WI, 1998, pp. 73–88. [2] N.J. Oldroyd, A.J. Urquhart, C.P. Kimpton, E.S. Millican, S.K. Watson, T. Downes, P.D. Gill, A highly discriminating octoplex short tandem repeat polymerase chain reaction system suitable for human individual identification, Electrophoresis 16 (1995) 334–337. [3] B. Budowle, T.R. Moretti, A.L. Baumstark, D.A. Defenbaugh, K.M. Keys, Population data on the thirteen CODIS core short tandem repeat loci in African Americans, US Caucasians, Hispanics, Bahamians, Jamaicans, and Trinidadians, Forensic Sci. 44 (1999) 1277–1286. [4] B. Budowle, A. Masibay, S.J. Anderson, C. Barna, L. Biega, S. Brenneke, B. Brown, J. Cramer, G.A. Degroot, D. Douglas, B. Duceman, A. Eastman, R. Giles, J. Hamill, D.W. Janssen, T.D. Kupferschmid, T. Lawton, C. Lemire, B. Llewellyn, T. Morretti, J. Neves, C. Palaski, S. Schueler, J. Sgueglia, C. Sprecher, C. Tomsey, D. Yet, STR primer concordance study, Forensic Sci. Int. 124 (2001) 47–54. [5] B. Budowle, D.A. Defenbaugh, K.M. Keys, Genetic cvariation at nine short tandem repeat loci in Chamorros and Filipinos from Guam, Legal Med. 2 (2000) 26–30. [6] P.S. Walsh, J. Varlaro, R. Reynolds, A rapid chemiluminescent method for quantitation of human DNA, Nucleic Acids Res. 20 (1992) 5061–5065. [7] K. Lazaruk, J. Wallin, C. Holt, T. Nguyen, P.S. Walsh, Sequence variation in humans and other primates at six short tandem repeat loci used in forensic identity testing, Forensic Sci. Int. 119 (2001) 1–10. [8] AmpF‘STR Identifiler User’s Manual, Version A, Applera Corporation, Foster City, 2001. [9] P. Collins, L. Hennessy, C. Leibelt, R. Roby, D. Reeder, P. Foxall, Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338, D19S433 and

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24]

227

Amelogenin: the AmpF‘STR1 IdentifilerTM PCR amplification kit, J. Forensic Sci. (2003) (accepted). M.C. Kline, B. Jenkins, S. Rodgers, Non-amplification of a vWA allele, J. Forensic Sci. 43 (1998) 250. S. Walsh, M.C. Kline, B. Jenkins, S. Rodgers, Nonamplification of a vWA allele, J. Forensic Sci. 43 (1998) 1103–1104. B. Budowle, STR allele concordance between different primer sets—a brief summary, Profiles DNA 3 (2000) 10–11. B. Budowle, C.J. Sprecher, Concordance study on population database samples using the PowerPlexTM 16 kit and AmpF‘STR1 Profiler PlusTM kit and AmpF‘STR1 CofilerTM kit, J. Forensic Sci. 46 (2001) 637–641. J. Wallin, C. Holt, K. Lazaruk, T. Nguyen, P.S. Walsh, Constructing universal multiplex PCR systems for comparative genotyping, J. Forensic Sci. 47 (2002) 1–14. M.S. Nelson, E.N. Levedakou, J.R. Matthews, B.E. Early, D.A. Freeman, C.A. Kuhn, C.J. Sprecher, A.S. Amin, K.C. McElfresh, J.W. Schumm, Detection of a primer-binding site polymorphism for the STR locus D16S539 using the Poerplex1 1.1 system and validation of a degenerate primer to correct for the polymorphism, J. Forensic Sci. 47 (2002) 345–349. L. Gusmao, A. Amorim, M.J. Prata, L. Pereira, M.V. Lareu, A. Carracedo, Failed PCR amplification of MBP-STR alleles due to polymorphism in the primer annealing region, Int. J. Legal Med. 108 (1996) 313–315. G.R. Han, E.S. Song, J.J. Hwang, Non-amplification of an allele of the D8S1179 locus due to a point mutation, Int. J. Legal Med. 115 (2001) 45–47. C. Alves, L. Gusmao, L. Pereira, A. Amorim, Multiplex STR genotyping: comparison study, population data and new sequence information, in: Proceedings of the 19th International Congress of the International Society for Forensic Genetics, Munster, Germany, 2001, p. 67. Quality assurance standards for forensic DNA testing laboratories, DNA Advisory Board, Federal Bureau of Investigation, US Department of Justice, 1998. N. Peyret, P.A. Seneviratne, H.T. Allawi, J. SantaLucia, Nearest-neighbor thermodynamics and NMR of DNA sequences with internal AA, CC, GG, and TT mismatches, Biochemistry 38 (1999) 3468–3477. A.T. Hatim, J. SantaLucia, Nearest-neighbor thermodynamics of Internal AC mismatches in DNA: sequence dependence and pH effects, Biochemistry 37 (1998) 9435–9444. A.T. Hatim, J. SantaLucia, Thermodynamics and NMR of internal GT mismatches in DNA, Biochemistry 36 (1997) 10581–10594. J.F. Williams, Optimization strategies for the polymerase chain reaction, BioTechniques 7 (1989) 762–768. R. Chakraborty, D.N. Stivers, B. Su, Y. Zhong, B. Budowle, The utility of short tandem repeat loci beyond human identification: implications for development of new DNA typing systems, Electrophoresis 20 (1999) 1682–1689.