Developmental Validation of the AmpFℓSTR^® Identifiler^® Plus PCR Amplification Kit: An Established Multiplex Assay with Improved Performance

Abstract

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing multiplex polymerase chain reaction (PCR) remains the primary method for genotyping forensic samples. The AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit is an improved version of the AmpFℓSTR(®) Identifiler(®) PCR Amplification Kit and amplifies the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant, amelogenin, and two internationally accepted loci, D2S1338 and D19S433. While the primer sequences and dye configurations were unchanged, the AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit features an enhanced buffer formulation and an optimized PCR cycling protocol that increases sensitivity, provides better tolerance to PCR inhibitors, and improves performance on mixture samples. The AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit has been validated according to the FBI/National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The validation results support the use of the AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit for human identity and parentage testing.

The effects of metal ion PCR inhibitors on results obtained with the Quantifiler ® Human DNA Quantification Kit

“…These analyses are conducted through the use of commercially-available kits, such as Identifiler 1 Plus (Applied Biosystems 1 , Foster City, CA) and the PowerPlex 1 16HS System (Promega Corporation, Madison, WI). These kits are designed to co-amplify targets of 15 polymorphic STR loci plus Amelogenin, a sex determining marker, and contain advanced buffering components designed to produce results in the presence of polymerase chain reaction (PCR) inhibitors [1,2]. Optimal performance of these kits requires adding DNA template to PCR reagents within the working range of the kit.…”

Challenging and Rare DNA Profiling of Burned Corpses at the State Forensic Science Laboratory of Jharkhand

“…AmpFISTR Identifiler Plus ® kit [8] was used to multiplex PCR reaction for co-amplification of 15 autosomal STRs loci and a gender locus (Table 2). Using 25 μl PCR amplification mixture (10.5 μl of PCR reaction mix, 5.5 μl of Identifier Plus ® Primer Set, 9.0 μl of nucleasefree water and 1 μl of DNA template), amplification was carried out under conditions of initial denaturation at 95°C for 10 min, followed by 29 cycles of denaturation at 94°C for 1 min, annealing at 59°C for 1 min and extension at 72°C for 1 min, and a final extension step at 60 °C for 60 min.…”

Crime investigation through DNA methylation analysis: methods and applications in forensics

“…These DMRs observed among multiple tissues (tDMRs) of humans depicts a molecular view of epigenetic changes marked among different tissues. Forensic cases involving monozygotic twins study, tissues identification, human age determination or predicting his/her behavior are not possible to assort through STR profiling which is solely based on variation in DNA sequence repeats called short tandem repeats (Oostdik et al, 2014;Wang et al, 2012). On the other hand, methylation marks flagged in the DNA sequence is a result of cell type as well as its immediate environment (Marsit, 2015).…”

“…Such obscure exhibits present a big challenge in front of forensic scientists to determine the actual cause/severity of the crime. The current method of DNA investigation in forensic laboratories is carried out by Identifiler®/GlobalFiler® Express kits (Life Science Technology Ltd) (Hennessy et al, 2014;Mulero et al, 2008;Wang et al, 2012) or PowerPlex® kit (Promega) (Oostdik et al, 2014) or Investigator® 24Plex kits (Qiagen) , which measures at least the 13 core short tandem repeats (STR) loci recommended by Federal Bureau of Investigation (FBI), USA. The method is quite fast and concludes all the polymorphic STR alleles by multiplex amplification of DNA from the picogram (pg) levels of DNA concentration (Green et al, 2013).…”