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.
Department of Biotechnology, KTH Royal Institute of Technology, Teknikringen 30, S-10044, Stockholm, Sweden.
J
J
Odeberg
A
A
Ahmadian
C
C
Williams
M
M
Uhlén
F
F
Pontén
J
J
Lundeberg
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