Fortunately, because STR loci can be amplified with fairly small product sizes, there is a greater chance for the STR primers to find some intact DNA strands for amplification. In addition, the narrow size range of STR alleles benefits analysis of degraded DNA samples because allele dropout via preferential amplification of the smaller allele is less likely to occur since both alleles in a heterozygous individual are similar in size.
A number of experiments have shown that there is an inverse relationship between the size of the locus and successful PCR amplification from degraded DNA samples, such as those obtained from a crime scene or a mass disaster (Whitaker et al. 1995, Sparkes et al. 1996, Takahashi et al. 1997,
(a) Agarose yield gel results
High molecular weight DNA in a tight band
Smear of degraded DNA fragments
(b) Degraded DNA sample
Good quality Degraded DNA DNA
Impact of degraded DNA on (a) agarose yield gel results and (b) STR typing. (a) Degraded DNA is broken up into small pieces that appear as a smear on a scanned yield gel compared to good quality DNA possessing intact high molecular weight DNA. (b) Signal strength is generally lost with larger size PCR products when STR typing is performed on degraded DNA, such as is shown from the green dye-labeled loci in the PowerPlex 16 kit. Thus, 180 bp D13S317 PCR products have a higher signal than 400 bp Penta D amplicons because more DNA molecules are intact in the 200 bp versus the 400 bp size range.
Schneider et al. 2004). The STR loci with larger sized amplicons in a multiplex amplification, such as D18S51 and FGA, are the first to drop out of the DNA profile when amplifying extremely degraded DNA samples (see Figure 7.1).
In one of the first studies demonstrating the value of multiplex STR analysis with degraded DNA samples, the Forensic Science Service was able to successfully type a majority of 73 duplicate pathological samples obtained from the Waco disaster with four STR markers (Whitaker et al. 1995). They observed no allele dropout and obtained concordant results on all samples where alleles were scored. A correlation was observed between successful typing at a locus and the average length of the alleles at that locus. The FES/FPS locus, which has alleles in the size range of 212-240 bp, only yielded 91 successful amplifications while the VWA locus with alleles ranging from 130-169 bp had 115 successful amplifications. Thus, loci with the larger alleles failed first. In addition, amelogenin amplicons (106 or 112 bp) were obtained on all 24 samples examined as part of the Waco identification program.
The potential for analysis of degraded DNA samples is an area where multiplex STR systems really shine over previously used DNA markers. STRs are more sensitive than single-locus probe RFLP methods, less prone to allelic dropout than VNTR systems (AmpFLPs) such as D1S80, and more discriminating than other PCR-based typing methods, such as HLA-DQA1 and AmpliType PolyMarker.
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