STR primer concordance study

Abstract

Over 1500 population database samples comprising African Americans, Caucasians, Hispanics, Native Americans, Chamorros and Filipinos were typed using the PowerPlex^® 16 and the Profiler Plus™/COfiler™ kits. Except for the D8S1179 locus in Chamorros and Filipinos from Guam, there were eight examples in which a typing difference due to allele dropout was observed. At the D8S1179 locus in the population samples from Guam, there were 13 examples of allele dropout observed when using the Profiler Plus kit. The data support that the primers used in the PowerPlex^® 16, Profiler Plus™, and COfiler™ kits are reliable for typing reference samples that are for use in CODIS. In addition, allele frequency databases have been established for the STR loci Penta D and Penta E. Both loci are highly polymorphic.

Introduction

United States forensic laboratories that contribute to the National DNA Index System database, utilizing the software known as combined DNA index system (CODIS), are required to type 13 short tandem repeat (STR) loci from DNA acquired from convicted felons. The STR loci are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11 [1]. To facilitate analysis of the core CODIS STR loci, commercial kits have been developed by two manufacturers (i.e. Applied Biosystems, Foster City, CA, and Promega Corp., Madison, WI). These kits contain primer sets for amplifying the various STR loci by the polymerase chain reaction (PCR). Primers are designed to anneal to invariant regions flanking the STR locus of interest. Regardless of how well designed, when a sufficiently large number of individuals are typed, it is likely that a variant will be seen in the “invariant” flanking region of the target sequence where a primer hybridizes. If a mismatch is proximal to the 3′ end of the primer, the mismatch may hinder or prevent primer extension during the PCR. When no product is synthesized, allele dropout results.

For a particular locus, the sequence of primers (and thus the primer binding sites) are presumably different from each manufacturer (and in some cases between different versions of kits from the same manufacturer). Within and between laboratories that use the same primer sets, generally there is little concern for profile comparisons about allele dropout due to primer mismatch; typically, multiple analyses of DNA samples from the same source would yield the same profile. Although in some rare casework scenarios where inhibitors or contaminants co-purify with the DNA, it may be possible to observe an allele dropout in an evidence sample and not in a reference sample from the same source using the same primer sets, and vice versa.

A comparison of typing results from samples that have been analyzed using the different primer sets (or kits) from the different manufacturers reveals the degree of allele dropout that may occur with a particular primer set. The results of such a comparison can be used as part of the evaluation process for the typing reliability of the primer sets.

In the current study, concordance results were compiled on samples typed using both the PowerPlex^® 16 kit (Promega Corp., Madison, WI) and the Profiler Plus™/COfiler™ kits (Applied Biosystems, Foster City, CA). The data show that for the major population groups (African American, Caucasian, and Hispanic) allele dropout is rare using either manufacturer’s primers.