Quantification of single nucleotide polymorphisms:: A novel method that combines primer extension assay and capillary electrophoresis

We present a novel method for accurate quantification of single nucleotide polymorphism (SNP) variants in transcripts and pooled DNAs in a one,tube reaction. Our approach is based on singled nucleotide primer extension (SNuPE) and laser induced fluorescence capillary electrophoresis (LIF-CE), and takes advantage of distinct mobilities of SNuPE products with different nucleotides incorporated at their 3' ends. The method, called SNuPE,ONCE, was tested on two polymorphisms and five mutations that comprised the three most frequent (similar to 70%) nucleotide changes in the human genome (C/T, A/G, and A/T). The usefulness of the method was demonstrated by analyzing nonsense-mediated mRNA instability in fibroblasts. Our data show 1) that the method provides highly reproducible relative allele frequencies (SD<0.017) with a good accuracy (e.g. for heterozygotes 0.500 +/- 0.036, P=0.01), depending on the sequence and the proportion of the SNP variants in the sample, and 2) that relative allele frequencies as low as 1% can be detected quantitatively and unambiguously. Our assay relies on a CE instrument available in many laboratories and offers a useful method for quantitative SNP genotyping as well as for a variety of expression studies. Hum Mutat 19:58-68, 2002. (C) 2001 Wiley-Liss, Inc.