Correction of Some Genotyping Errors in Automated Fluorescent Microsatellite Analysis by Enzymatic Removal of One Base Overhangs

Abstract

The conjunction of high resolution genetic maps based on (CA)n microsatellite markers (1) and fluorescent genotyping (2) has led to research programs which require the determination of hundreds of thousands of genotypes. However, the migration profile of a (CA)n microsatellite marker after PCR (polymerase chain reaction) is often complicated because of slippage of Taq polymerase during PCR, and because of the ability of this polymerase to add an extra base at the end of the amplified fragments in a template dependent manner (3). In the traces generated by a fluorescent sequencer, these phenomena generate spurious bands as shown in Figure 1. These extra bands are usually referred to as ‘shadow bands’ (4). This complicated pattern is relatively easily recognized by an experienced eye, but may mislead the software which automatically determines the sample alleles (we use ABI 373A sequencers with 24 cm gels for the electrophoresis, and the ABI software Genescan and Genotyper for peak sizing and allele calling respectively). Because of this, the operator must check visually all the traces and the allele calls made by the software. This is time-consuming, and prone to human error after a few hundred checks. We attempted therefore to eliminate most of the allele calling errors by eliminating the extra bases added by the Taq polymerase. We tried to do this by using Pfu polymerase either during or after PCR, or by using T4 DNA polymerase after PCR. Pfu polymerase is thermostable and possesses a proofreading activity (3). In some cases, however, the extra base added by the Taq polymerase was not completely removed by this enzyme (Fig. 2A and B). This phenomenon is probably due to competition between the extendase activity of the Taq polymerase and the exonuclease activity of the Pfu polymerase as both enzymes are active at 72 C. On the contrary, and as suggested by others (5), a T4 polymerase treatment, similar to that conventionally used to make blunt double-stranded linear DNA fragments, completely removed the bases added by the Taq polymerase (without exception so far; Fig. 2C). The simplification of the electrophoresis pattern is sometimes spectacular (Fig. 3). The use of Pfu polymerase alone during PCR avoids the problem of the extra base addition (3), and was used successfully in fluorescent genotyping (6). However, PCR efficiency is lower than when using Taq polymerase, making it necessary to precipitate the samples during multiplex analysis (data not shown). Figure 1. Typical electrophoresis pattern of a (CA)n microsatellite marker with shadow bands. The two alleles of this heterozygous sample are eight bases apart, and are shown with their estimated size. The rightmost peak of each group corresponds to an extra base addition (peaks labelled +1). On the left, we see peaks corresponding to slippage of the Taq polymerase (labelled –2 and 4), and the ‘+l peaks’ of these ‘slippage peaks’. For this marker, the normal peaks and the +l peaks have a similar height. That means that, depending on the samples, the Genotyper software will choose one peak or the other for the allele, leading to errors in the genetic data.