Ad. Greenwood et Dt. Burke, SINGLE NUCLEOTIDE PRIMER EXTENSION - QUANTITATIVE RANGE, VARIABILITY,AND MULTIPLEX ANALYSIS, PCR methods and applications, 6(4), 1996, pp. 336-348
The quantitative measurement of transcription products from homologous
alleles at a diploid locus has broad application for the study of mam
malian gene expression. Single nucleotide primer extension (SNuPE) ana
lysis is a simple and sensitive method for allelic transcript discrimi
nation requiring only 1 bp difference between alleles. In this study t
he effective range, experimental variation, and the influences of poly
(dT)-primed and gene-specific reverse transcriptions are characterized
. The ability to analyze several genes fron 1 a single reverse transcr
iption reaction is assessed as well. For the genes examined, the maxim
um range of detection is reached when the minor transcript represents
1/250 of the major allele. Relatively little error is seen within or b
etween assays and linearity of response is maintained over an approxim
ately thousandfold range.