Preferential PCR amplification of alleles: mechanisms and solutions.

Abstract

The preferential PCR amplification of one allele relative to another in a heterozygous sample could result in an incorrect or ambiguous genetic typing of that sample. There are several mechanisms that could potentially lead to such preferential PCR amplification. First, preferential amplification can result from significant GC% differences be-

Inhibition of DNA synthesis by K+‐stabilised G‐quadruplex promotes allelic preferential amplification

“…containing the (TGGGGC) 4 motif Why is the longest allele disfavoured during PCR amplification in the presence of K þ ? To address this question, we sequenced both alleles of the individual 01.…”

“…For G-quadruplex formation, 280 fmol of each end-labelled oligonucleotide was incubated at 95°C for 5 min in 200 ll of TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) with 50 or 100 mM KCl. Incubations were also performed in TE buffer with 100 mM NaCl, LiCl or NH 4 Cl instead of KCl. After cooling down to room temperature, the DNAs were held for 60 min at 37°C during 60 min prior to dimethylsulfate (DMS) treatment.…”

“…To analyse the inhibition of DNA synthesis provoked by the (TGGGGC) 4 motif, we carried out a DNA sequence assay with and without KCl. The sequencing reactions were performed following the protocol of the ABI PRISM sequencing kit (Applied Biosystems).…”

“…This effect may lead to poor reliability and misdiagnosis in genetic analysis. The causes of this artefact are not fully understood, but some that have been proposed include differences in allele length or GC richness, small amount of template producing stochastic fluctuations in the number of copies for each allele, and the existence of polymorphisms producing mismatches between the primer and its target sequence [4]. More complex mechanisms such as reannealing of complementary template (complete or truncated) and subsequent PCR clamping were also proposed to be involved in preferential amplification [5].…”

“…Currently, the analysis of minisatellites is performed by PCR amplification. However, with this technique there may occur artefacts such as slippage products [2], heteroduplexes [3], allelic drop-out and preferential amplification [4]. The latter artefact consists of a weak amplification of one allele in a heterozygous individual.…”