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

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Abstract
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 single‐ 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 (∼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 (SDP = 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.
Funding Information
  • Swiss Marfan Foundation
  • Telethon
  • Swiss National Science Foundation (SNF 32-59445.99, SNF 4037-055165)