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

Hum Mutat. 2002 Jan;19(1):58-68. doi: 10.1002/humu.10013.


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 ( approximately 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • B-Lymphocytes
  • Base Sequence / genetics
  • Cell Line, Transformed
  • Cell Transformation, Viral
  • Chromosome Mapping / methods
  • DNA Primers / genetics*
  • Electrophoresis, Capillary / methods*
  • Fibroblasts
  • Herpesvirus 4, Human
  • Humans
  • Molecular Sequence Data
  • Mutation, Missense / genetics
  • Polymorphism, Single Nucleotide / genetics*
  • RNA / genetics


  • DNA Primers
  • RNA