An amplification and ligation-based method to scan for unknown mutations in DNA.
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.
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.
Yuzhi
Y
Zhang
Manjit
M
Kaur
Brendan D
BD
Price
Sotirios
S
Tetradis
G Mike
GM
Makrigiorgos
D000225:Adenine; D034961:Antigens, Nuclear; D064007:Ataxia Telangiectasia Mutated Proteins; D017122:Bacteriophage T4 / Q000201:enzymology; D020137:Base Pair Mismatch / Q000235:genetics; D018797:Cell Cycle Proteins; D004265:DNA Helicases; D011088:DNA Ligases; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics; D006147:Guanine; D006801:Humans; D000072200:Ku Autoantigen; D008025:Ligases; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011130:RNA Ligase (ATP); D025521:Tumor Suppressor Proteins; D014764:Viral Proteins
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes; D025521:Tumor Suppressor Proteins; D014764:Viral Proteins; D006147:Guanine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D008025:Ligases; D011088:DNA Ligases; D011130:RNA Ligase (ATP); D000225:Adenine