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 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.
Mátyás
Gábor
G
University Children's Hospital, Division of Metabolism and Molecular Pediatrics, Zurich, Switzerland. gabor.matyas@kispi.unizh.ch
Giunta
Cecilia
C
Steinmann
Beat
B
Hossle
Johann Peter
JP
Hellwig
Robert
R
D000483:Alleles; D001402:B-Lymphocytes; D001483:Base Sequence / Q000235:genetics; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002874:Chromosome Mapping / Q000379:methods; D017931:DNA Primers / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005347:Fibroblasts; D004854:Herpesvirus 4, Human; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012313:RNA / Q000235:genetics
D017931:DNA Primers; D012313:RNA