Identification of coding single nucleotide polymorphisms and mutations by combination of genome tiling arrays and enrichment/depletion of mismatch cDNAs.
Genome tiling array technology combined with a method for both enrichment and depletion of mismatch-containing cDNA fragments offers a useful approach for detecting coding single nucleotide polymorphisms (cSNPs) and mutations in pooled cDNA samples. Enriched mismatch and perfect match cDNA samples from human primary melanoma cells and normal melanocytes were obtained by selection using mismatch repair thymine DNA glycosylase-bound beads. These cDNA samples were then labeled and hybridized to Encyclopedia of DNA Elements genome tiling arrays. The results revealed that the hybridization intensity values of potential cDNA variation regions of the enriched mismatch samples increased, whereas the hybridization intensity values of corresponding regions of the enriched perfect match samples decreased. Six potential mutations were confirmed by polymerase chain reaction product sequencing, including two novel heterozygous mutations in melanoma cells. We suggest that this strategy should increase the efficiency of both cSNP and mutation detection throughout the entire human genome and decrease the cost and complexity of genomewide analysis of cDNA variations.
Liu
Meng-Min
MM
Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA. meng-min.liu@yale.edu
Weissman
Sherman M
SM
Tang
Ling
L
D020137:Base Pair Mismatch; D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005821:Genetic Techniques; D006801:Humans; D008544:Melanocytes / Q000378:metabolism; D008545:Melanoma / Q000235:genetics; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D004273:DNA, Neoplasm