Strategies for genotyping: Effectiveness of tailing primers to increase accuracy in short tandem repeat...

A problem associated with automated analysis of fluorescently labeled fragments separated by slab gel or capillary electrophoresis is the doublet peak formed when Taq DNA Polymerase adds a nontemplated nucleotide (generally an adenosine) to the 3' end of the product.This nontemplated addition (plus A) is primarily dependent on the 5' sequence of the reverse primer and, to a lesser extent, polymerase chain reaction (PCR) conditions. Primers may amplify the true product, the plus A product or a doublet product comprised of both. When using markers based on dinucleotide repeats, this single base pair difference can make binning and accurate automated analysis problematic. To drive the PCR reaction consistently to the plus A product, the sequence of the nonfluorescent primer used in amplification can be modified by adding a 5' tail favoring the nontemplated addition. The present study, conducted by the Fragment Analysis Research Group (FARG) of the Association of Biomolecular Resource Facilities, provided researchers with an opportunity to compare normal products amplified with a dinucleotide marker to products amplified with the same primer to which a 5' tail designed to promote the plus A product had been added. The study also included a sample amplified with a tetranucleotide repeat marker for comparison. The results from this study were returned to the FARG for comprehensive analysis and are reported here.