Accurate STR Allele Designations at the FGA and vWA Loci Despite Primer Site Polymorphisms

Abstract

Polymerase chain reaction (PCR)-based STR DNA typing systems are used extensively in the field of human identification. Under optimal PCR conditions, the amplicon yield from both alleles of an STR locus is expected to be approximately equivalent. However, it is reasonable to expect that rare genomic sequence polymorphisms will co-localize with well-designed primer sets and induce allele imbalance or "dropouts". Two samples were identified in the course of genotyping thousands of individuals with AmpF/STR R Profiler Plus TM that showed strong disparity in amplitude peak height of heterozygous peaks at the loci vWA and FGA. These samples were reamplified at reduced annealing temperature in an attempt to balance the peak heights. Nucleotide sequencing documented polymorphisms at the PCR primer binding sites of the affected alleles. The results indicate that reducing the annealing temperature to improve primer-binding efficiency at the mismatch and employing an alternative multiplex enhanced the data from both samples. Reducing annealing temperatures could provide a simple general solution to improving data quality for samples where polymorphisms are suspected to cause allele imbalance. Finally, we report on additional polymorphisms surrounding the vWA locus in a genetically diverse population.

Allelic drop-out in the STR system ACTBP2 (SE33) as a result of mutations in the primer binding region

“…Nevertheless, these mutations led to a complete failure of amplification and thereby to a false homozygote result, at least in a multiplex reaction. A similar phenomenon is already known from mutations in the primer binding regions of other STRs [7,8,9,10,11,12].…”

A D19S433 Primer Binding Site Mutation and the Frequency in Japanese of the Silent Allele It Causes

“…This indicated that the allele with 13 repeats was amplified with the silent allele-specific reverse primer of the ASP set. Sample G was typed as a homozygote (13,13) using the Identifiler Ò kit and the COM set. If sample G had contained the silent allele with the 13 repeat units, two peaks at a spacing of about three bases should have been observed in the ASP electropherogram profile.…”

“…Silent alleles caused by mutations at a primer binding site have been identified at several STR loci included in the Identifiler Ò kit (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). In most studies, the presence of a silent allele in an individual has been recognized by observing the discordant genotyping results of an individual after using the AmpF'STR Ò and PowerPlex 16 Ò kits (Promega).…”

Assessing quantitative chimerism longitudinally: technical considerations, clinical applications and routine feasibility

“…Such errors in estimating DNA quantity may independently develop because of intrinsic properties of markers included in the commercial multiplex kits. 38 Table 2 illustrates the occasional 5-10% variability in the %Chm values that can be seen among the 10 multiplex markers that we run routinely. Each sample represents a run on a separate day.…”