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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.

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 Ò 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.

The AmpFℓSTR(®) Identifiler(®) Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA(®) card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay's sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA(®) cards, and the assay's specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA(®) substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler(®) Direct Kit for forensic standards and database samples genotyping.

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