Julio J. Mulero

Development and Validation of the AmpFℓSTR® Yfiler™ PCR Amplification Kit: A Male Specific, Single Amplification 17 Y-STR Multiplex System*

ABSTRACT: In the past 5 years, there has been a substantial increase in the use of Y‐short tandem repeat loci (Y‐STRs) in forensic laboratories, especially in cases where typing autosomal STRs has met with limited success. The AmpFℓSTR® Yfiler™ PCR amplification kit simultaneously amplifies 17 Y‐STR loci including the loci in the “European minimal haplotype” (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393), the Scientific Working Group on DNA Analysis Methods (SWGDAM) recommended Y‐STR loci (DYS438 and DYS439), and the highly polymorphic loci DYS437, DYS448, DYS456, DYS458, Y GATA H4, and DYS635 (formerly known as Y GATA C4). The Yfiler™ kit was validated according to the FBI/National Standards and SWGDAM guidelines. Our results showed that full profiles are attainable with low levels of male DNA (below 125 pg) and that under optimized conditions, no detectable cross‐reactive products were obtained on human female DNA, bacteria, and commonly encountered animal species. Additionally, we demonstrated the ability to detect male specific profiles in admixed male and female blood samples at a ratio of 1:1000.

Development and Validation of the AmpFℓSTR® MiniFilerTM PCR Amplification Kit: A MiniSTR Multiplex for the Analysis of Degraded and/or PCR Inhibited DNA

Abstract:  DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFℓSTR® MiniFiler™ PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFℓSTR® Identifiler® PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFℓSTR® SGM Plus® kit. This informative nine‐locus multiplex, which includes the gender‐identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFℓSTR® Identifiler® and SGM Plus® kits. These data support that the MiniFiler™ kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.

Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits.

Abstract:  The AmpFℓSTR® MiniFilerTM polymerase chain reaction amplification kit developed by Applied Biosystems enables size reduction on eight of the larger STR loci amplified in the Identifiler® kit, which will aid recovery of information from highly degraded DNA samples. The MiniFilerTM Kit amplifies CSF1PO, FGA, D2S1338, D7S820, D13S317, D16S539, D18S51, and D21S11 as well as the sex‐typing locus amelogenin. A total of 1308 samples were evaluated with both the MiniFilerTM and Identifiler® STR kits: 449 African American, 445 Caucasian, 207 Hispanic, and 207 Asian individuals. Full concordance between Identifiler and MiniFiler Kits was observed in 99.7% (10,437 out of 10,464) STR allele calls compared. The 27 differences seen are listed in Table 1 and encompass the loci D13S317 (n = 14) and D16S539 (n = 10) as well as D18S51 (n = 1), D7S820 (n = 1), and CSF1PO (n = 1). Genotyping discrepancies between the Identifiler and MiniFiler kits were confirmed by reamplification of the samples and further testing using the PowerPlex® 16 kit in many cases. DNA sequence analysis was also performed in order to understand the nature of the genetic variations causing the allele dropout or apparent repeat unit shift.

Letter to the editor--nomenclature and allele repeat structure update for the Y-STR locus GATA H4.

Sir: Establishing a consensus nomenclature can facilitate data comparison for proficiency testing, quality assurance, and casework results. Efforts into nomenclature standardization should be supported and lauded. The DNA Commission of the International Society of Forensic Genetics (ISFG) has issued new recommendations on the nomenclature and specifically addressed the Y GATA H4 marker (1). There are differences in allele designations at the GATA H4 marker between those recommended in the Applied Biosystems AmpFlSTR Yfiler polymerase chain reaction amplification kit (Applied Biosystems, Foster City, CA) and the ISFG recommendations. The nomenclature for the GATA H4 marker in the Yfiler kit is based on the allele repeat structure defined by the National Institute of Standards and Technology Standard reference material (SRM) 2395 and the work of Butler et al. (2). In the Yfiler kit the variable core repeat (TAGA) is used to designate the alleles (such as alleles 8–13 in the allelic ladder of the Yfiler kit) (3,4). Furthermore, the Yfiler kit primers amplify the region now designated as the GATA H4.1 locus as defined by Gusmao et al. (5) and recommended by the ISFG Commission. The GATA H4.1 locus structure consists of a core repeat region designated as AGAT and nonvariable tetranucleotide repeats ((AGAT)4CTAT(AGAT)2(AGGT)3(AGAT)n) that are considered for allele number designation under the ISFG recommendations. Thus, there is a difference in allele nomenclature that depends on whether or not the nonvariable region is included. Those who choose to follow the allele nomenclature recommendations of the ISFG Commission should add a correction factor of nine to the Yfiler allele number, and they should refer to this marker as GATA H4.1. Employing the ISFG proposed allele designation for GATA H4.1 changes the Yfiler kit allelic ladder range from 8–13 to 17–22. Alternatively, those who amplify the entire GATA H4 region (GATA H4.1 and GATA H4.2) should add a correction factor of 16 to the Yfiler kit allele number. In this case, the Yfiler kit allelic ladder for GATA H4 ranges from 24 to 29.

Characterization of the N+3 Stutter Product in the Trinucleotide Repeat Locus DYS392

ABSTRACT: Stutter products generated during DNA amplification by the polymerase chain reaction (PCR) may complicate mixture interpretation. The PCR amplification of the DYS392 locus typically results in three distinct detectable PCR products: the true allele product (N), a stutter product three bases smaller (N−3), and a reproducible low‐level product, three bases larger (N+3). Sequence analysis of the N+3 product demonstrated that its sequence is one TAT repeat longer than the true allele product. Our experiments demonstrated that the quantity of both N−3 and N+3 stutter increased as the allele number increased. The percent stutter also increased as the magnesium concentration was increased in the reaction, as well as when the amount of input DNA was decreased. As both stutter products behave in a similar and reproducible fashion, the same rules that apply to the interpretation of N−3 stutter products in short tandem repeat analysis, can be applied to N+3 stutters. The characterization of the DYS392 N+3 product is the first detailed published study of a stutter product larger than the true allele.

Identification of a Novel Polymorphism in the X‐Chromosome Region Homologous to the DYS456 Locus

ABSTRACT: During an extensive multipopulation study with Y‐short tandem repeat (STR) loci, amplified using the AmpFℓSTR® Yfiler™ PCR amplification kit, amplification of a 71 bp fragment was observed in 2.32% of the male samples analyzed (N=3141). By direct sequencing of this fragment, it was determined that the primer binding sequences were identical to those of the DYS456 locus. A T to G single‐nucleotide polymorphism (SNP) enabled amplification of the 71 bp fragment. The SNP is located within an X–Y homologous region at Xq21.31 and was observed with the highest frequency within the African American and Sub‐Saharan African populations in our study. Presence of SNP on the X chromosome did not interfere with the reliability of typing the DYS456 locus and the other Y‐STR loci typeable using the AmpFℓSTR® Yfiler™ PCR amplification kit. Full profiles in a mixture of male:female at 1:4000 were obtained using the current configuration of the AmpFℓSTR Yfiler kit even in the presence of female DNA containing the G variant.