An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue.
Knowledge of inherited and sporadic mutations in known and candidate cancer genes may influence clinical decisions. We have developed a mutation scanning method that combines thermostable EndonucleaseV (Endo V) and DNA ligase. Variant and wild-type PCR amplicons are generated using fluorescently labeled primers, and heteroduplexed. Thermotoga maritima (Tma) EndoV recognizes and primarily cleaves heteroduplex DNA one base 3' to the mismatch, as well as nicking matched DNA at low levels. Thermus species (Tsp.) AK16D DNA ligase reseals the background nicks to create a highly sensitive and specific assay. The fragment mobility on a DNA sequencing gel reveals the approximate position of the mutation. This method identified 31/35 and 8/8 unique point mutations and insertions/deletions, respectively, in the p53, VHL, K-ras, APC, BRCA1, and BRCA2 genes. The method has the sensitivity to detect K-ras mutations diluted 1 : 20 with wild-type DNA, a p53 mutation in a 1.7 kb amplicon, and unknown p53 mutations in pooled DNA samples. EndoV/Ligase mutation scanning combined with PCR/LDR/Universal array proved superior to automated DNA sequencing for detecting p53 mutations in colon tumors. This technique is well suited for scanning low-frequency mutations in pooled samples and for analysing tumor DNA containing a minority of the unknown mutation.
Huang
Jianmin
J
Department of Microbiology, Box 62, Hearst Microbiology Research Center, Strang Cancer Prevention Center, Joan and Sanford I Weill Medical College of Cornell University, Room B-406, 1300 York Avenue, New York, NY 10021, USA.
Kirk
Brian
B
Favis
Reyna
R
Soussi
Thierry
T
Paty
Philip
P
Cao
Weiguo
W
Barany
Francis
F
D000230:Adenocarcinoma / Q000235:genetics*; D001483:Base Sequence; D015374:Biosensing Techniques; D015179:Colorectal Neoplasms / Q000235:genetics*; D011088:DNA Ligases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000235:genetics*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D017353:Gene Deletion; D016147:Genes, Tumor Suppressor / Q000502:physiology*; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004273:DNA, Neoplasm; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); D011088:DNA Ligases