PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.
The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.
Division of Human Genetics, Medical University Innsbruck, Peter-Mayr-Strasse 1, Innsbruck, 6020, Austria, katharina.wimmer@i-med.ac.at.
Katharina
K
Wimmer
Annekatrin
A
Wernstedt
D000251:Adenosine Triphosphatases / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D045643:DNA Repair Enzymes / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004843:Epistasis, Genetic; D006801:Humans; D000070976:Mismatch Repair Endonuclease PMS2; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011544:Pseudogenes / Q000235:genetics*; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D045643:DNA Repair Enzymes