Context-dependent Taq-polymerase-mediated nucleotide alterations, as revealed by direct sequencing of the ZNF189 gene: implications for mutation detection.
We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.
Odeberg
J
J
Department of Biotechnology, KTH Royal Institute of Technology, Teknikringen 30, S-10044, Stockholm, Sweden.
Ahmadian
A
A
Williams
C
C
Uhlén
M
M
Pontén
F
F
Lundeberg
J
J
D016477:Artifacts; D001483:Base Sequence; D002280:Basal Cell Carcinoma / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D002899:Chromosomes, Human, Pair 9 / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics; D019656:Loss of Heterozygosity; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism*; D013698:Templates, Genetic
D004268:DNA-Binding Proteins; D009711:Nucleotides; C114474:ZNF189 protein, human; D019914:Taq Polymerase