Simultaneous quantitative and allele-specific expression analysis with real competitive PCR

Background: For a diploid organism such as human, the two alleles
of a particular gene can be expressed at different levels due to X
chromosome inactivation, gene imprinting, different local promoter
activity, or mRNA stability. Recently, imbalanced allelic
expression was found to be common in human and can follow Mendelian
inheritance. Here we present a method that employs real competitive
PCR for allele-specific expression analysis. Results: A transcribed
mutation such as a single nucleotide polymorphism ( SNP) is used as
the marker for allele-specific expression analysis. A synthetic
mutation created in the competitor is close to a natural mutation
site in the cDNA sequence. PCR is used to amplify the two cDNA
sequences from the two alleles and the competitor. A base extension
reaction with a mixture of ddNTPs/ dNTP is used to generate three
oligonucleotides for the two cDNAs and the competitor. The three
products are identified and their ratios are calculated based on
their peak areas in the MALDI-TOF mass spectrum. Several examples
are given to illustrate how allele-specific gene expression can be
applied in different biological studies. Conclusions: This
technique can quantify the absolute expression level of each
individual allele of a gene with high precision and throughput.