Single nucleotide primer extension: quantitative range, variability, and multiplex analysis.
The quantitative measurement of transcription products from homologous alleles at a diploid locus has broad application for the study of mammalian gene expression. Single nucleotide primer extension (SNuPE) analysis is a simple and sensitive method for allelic transcript discrimination requiring only 1 bp difference between alleles. In this study the effective range, experimental variation, and the influences of poly(dT)-primed and gene-specific reverse transcriptions are characterized. The ability to analyze several genes from a single reverse transcription reaction is assessed as well. For the genes examined, the maximum range of detection is reached when the minor transcript represents 1/250 of the major allele. Relatively little error is seen within or between assays and linearity of response is maintained over an approximately thousandfold range.
Greenwood
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Department of Human Genetics, University of Michigan, Ann Arbor 48105-0618, USA.
Burke
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DT
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007706:Menkes Kinky Hair Syndrome / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000235:genetics; D011071:Poly T / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012260:Ribonucleases / Q000378:metabolism; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014158:Transcription, Genetic
D017931:DNA Primers; D011071:Poly T; D009954:Ornithine Carbamoyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D012260:Ribonucleases