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Optimization of the D8S1179, D21S11 and D18S51 STR Loci for Incorporation into AmpFlSTRÔ Green II and AmpFlSTR Profiler IIÔ

The STR loci D8S1179, D21S11 and D18S51 have been combined for co-amplification in the AmpFlSTRÔ Green II kit. Extensive primer modification from the published sequences was necessary to achieve good size separation between the loci (all 1abeled with JOE NHS-ester dye), robust amplification (±2°C allowable for all thermal cycler parameters), peak height balance between loci, specific amplification and maximal non-template-dependent A addition to all PCR products. The D8S1179 primers have been modified to yield a smaller size PCR product which will not overlap with the D21S11 allelic products; to allow for more robust amplification at annealing temperatures of up to 61°C, and to minimize the problematic ±A split peaks seen when using the published primer sequences. The D21S11 primers were modified to increase overall product yield and to increase the spacing between the D21S11 and D18S51 allele sizes. The D18S51 primers were modified to provide improved amplification specificity under conditions of lower stringency, i.e. more difficult DNA samples. All of these modifications have produced a multiplex kit that is extremely robust when used with the optimized AmpFISTRÔ PCR Reaction Mix and AmpliTaq Gold^® DNA Polymerase.

D21S11 is a more complex STR locus with many 2 bp, and some 1 bp variant alleles in most populations. The higher frequency of these 2 bp variant alleles has necessitated the incorporation of these 2 bp alleles into the D21S11 allelic ladder, and therefore the ability of detection systems to resolve 1 and 2 bp alleles becomes more important for this locus. The ABI PRISMÔ 310 Genetic Analyzer and 377 DNA Sequencer, in conjunction with GeneScan^® 2.1 and Genotyper^® 2.0 software, both provide the ability to resolve and type these variant alleles.

The D8S1179, D21S11, and D18S51 1oci are included the in the AmpFlSTR Profiler IIÔ kit, along with the Blue and Yellow loci from the AmpFISTR ProfilerÔ kit. This combination of loci provides a PD approximately 1 in 70 billion. 200 Caucasian and 195 African-American population samples have been typed and many interesting alleles from the Green II loci have been sequenced in both the primer binding region and the tetranucleotide repeat region. In addition, TWGDAM validation experiments including: non-probative case samples, mixed specimens, samples deposited on a variety of difficult substrates, degraded DNA, and samples containing PCR inhibitors have been analyzed.

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