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Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.

In the past decade, fluorescence-based DNA detection systems have been widely used in forensic DNA analysis. Fluorescence detection methods have greatly aided the sensitivity and ease of measurement of PCR amplified short tandem repeat (STR) alleles. In the new multiplexed STR genotyping kits, fluorescent dyes are covalently coupled to primers for each locus. Fluorescence measurements involve detecting light emitted from an excited dye at a specific wavelength. Filters are used to select fluorescent dye signals at optimal wavelength ranges. In ABI PRISMR instruments, fluorescent signals are separated by a diffraction grating and projected onto a chargecoupled device (CCD) camera during data collection. Multicomponent analysis is performed using a matrix that excludes the contribution of neighboring dyes. Earlier kits from Applied Biosytems (AmpFlSTRRkits) used PCR primers with NHS-ester dyes 5-FAMk, JOEk or NEDk and ROXk dyes and emission spectra ranging from 522 to 607 nm. More recently, Applied Biosystems has developed a unique 5-dye technology for automated DNA fragment analysis. The introduction of the new 5-dye chemistry involves replacement of 5-FAM with 6-FAMk, JOE with VICk and ROX with LIZk and incorporation of the new PETk dye into the existing system. The five dyes, 6-FAM, VIC, NED, PET and LIZ, expand the spectral detection range on the ABI PRISM instrumentation to 660 nm. The addition of more dyes thus enables more loci to be multiplexed into a single PCR amplification. Additionally, the new dyes will increase genotyping throughput significantly since more loci can be analyzed simultaneously in a single lane or capillary. One advantage of this arrangement is that minimal hardware changes to existing instrument platforms is necessary.