A robust method for detecting CHK2/RAD53 mutations in genomic DNA.
While screening for germline CHK2 mutations in cancer cases by heteroduplex CSGE, we observed that additional PCR fragments were generated from the 3' end region of the gene that includes exons 11-14. Direct sequencing of these fragments suggested that homologous loci (possibly pseudogenes) were concomitantly being amplified. Searches of public sequence databases showed that a number of areas of the genome show a high degree of homology to exons 10-14 of the CHK2 gene. The presence of these homologous regions means that standard screening methods for detecting mutations in CHK2, based on PCR of genomic DNA, are prone to error. To circumvent this problem, we have developed a strategy, based on long-range PCR, to screen the functional copy of CHK2. Using this approach it is possible to carry out a comprehensive mutational analysis of CHK2 from genomic DNA.
Sodha
Nayanta
N
Royal Marsden NHS Trust, Surrey, UK. nayanta@icr.ac.uk
Houlston
Richard S
RS
Williams
Richard
R
Yuille
Martin A
MA
Mangion
John
J
Eeles
Rosalind A
RA
D001483:Base Sequence; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D030541:Databases, Genetic; D005091:Exons / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D018095:Germ-Line Mutation / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011544:Pseudogenes / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D018797:Cell Cycle Proteins; D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae