Chien-Wei Chang

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.

Development and Validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: A Multiplex Assay for the Direct Amplification of Single‐Source Samples*,†

Abstract:  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.

Characterization of 114 insertion/deletion (INDEL) polymorphisms, and selection for a global INDEL panel for human identification.

Bi-Allelic Insertions and Deletions (INDELs) are a powerful set of genetic markers for Human Identification (HID). They have certain desirable features, such as low mutation rates, no stutter, and potentially small amplicon sizes that could prove effective in some circumstances. In this study, we analyzed the distribution of 114 INDELs in four North American populations (Caucasian, African American, Southwest Hispanic, and Asian) to estimate their distribution in major global populations. Of the 114 INDELs a primary panel of 38 candidate markers was selected that met the criteria of (1) a minimum allele frequency of greater than 0.20 across the populations studied; (2) general concordance with Hardy-Weinberg equilibrium (HWE) expectations; (3) relatively low FST based on the major populations; (4) physical distance between markers greater than 40 Mbp; and (5) a lack of linkage disequilibria between syntenic markers. Additionally, another 11 supplemental markers were selected for an expanded panel of 49 markers which met the above criteria, with the exception that they are separated at least by 20 Mbp. The resulting panels had Random Match Probabilities that were at least 10(-16) and 10(-19), respectively, and combined FST values of approximately 0.02. Given these findings, these INDELs should be useful for HID.