Rapid genotyping of plant samples using TaqMan TM assays

Abstract

Abstract Insertional mutagenesis is a routine method used in reverse genetics, but commonly-used end-point PCR molecular screening methods are flawed because they rely on negative results and require labor intensive gDNA extractions. To address this, we have developed TaqMan™ genotyping assays that allow us to compare the copy number of specific alleles of a gene of interest to an internal control. The method is effective with ‘on-the-fly’ template prep with no purification that allows for streamlined genotyping of up to 95 samples in ~ 2 hours. TaqMan™ genotyping has been effective in both identifying all possible genotypes (wild type, heterozygous, and homozygous) for insertional mutants and for determining transgene copy numbers of individual transformation events in transgenic lines. Expression of maize transposon-insertion alleles was determined and the correlation between the transposon’s insertion location and its ability to effect gene expression was examined.