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An amplification and ligation‐based method to scan for unknown mutations in DNA
Author(s) -
Zhang Yuzhi,
Kaur Manjit,
Price Brendan D.,
Tetradis Sotirios,
Makrigiorgos G. Mike
Publication year - 2002
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.10106
Subject(s) - biology , sequencing by ligation , microbiology and biotechnology , oligonucleotide , dna , ap site , point mutation , dna ligase , dna glycosylase , genomic dna , gene , dna polymerase , mutation , dna repair , genetics , genomic library , base sequence
A new approach is presented for the sensitive and selective scanning for unknown DNA mutations, based on ligation‐mediated PCR and the use of the glycosylases TDG and MutY. These two highly selective enzymes together can detect about 70% of commonly observed polymorphisms and mutations in human tumors. DNA is cross‐hybridized to form mismatches at the positions of point mutations, de‐phosphorylated to eliminate any pre‐existing phosphorylated DNA ends, and then exposed to enzymatic treatment to remove mismatched thymidine (TDG) or adenine (MutY). The resulting apurinic/apyrimidinic sites at the position of the mismatches are heat‐converted to 5′‐phosphate‐containing strand breaks, the DNA is denatured, and an oligonucleotide is ligated at the position of the newly created 5′‐phosphate‐containing DNA ends. The ligated oligonucleotide then participates in a PCR reaction that amplifies exponentially only the mutation‐containing fragments. Using this method, A→G mutations in a p53 (TP53)‐containing system, T→G, G→A, and C→A, mutations in the Ku gene (XRCC5), and ATM, gene for a number of patient‐derived genomic DNA samples have been successfully screened. This PCR‐based assay is capable of detecting one mutated allele in 100 normal alleles and requires 5 to 100 ng of genomic DNA as starting material. The assay allows final visualization of the mutated fragments on a common ethidium gel or biotinylation and use in a capture format, potentially allowing the isolation of diverse mutated DNA fragments simultaneously. This versatile new approach should allow high throughput detection of DNA alterations and application in diverse areas of human mutation research. © 2002 Wiley‐Liss, Inc.