An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue

Abstract

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.

Enzymatic Mutation Detection Technologies

“…Similarly, the nicking and rejoining activities of topoisomerases were exploited in mutation detection, but this method suffers from nonspecific cuts in the DNA and the formation of covalent protein-DNA complexes (Reference 27); A.T. Yeung, unpublished data). Recently, an improved nicking-ligation system based on endonuclease V and a long repair step by thermostable DNA replication ligase has been devised (28,29). …”

Endonucleolytic mutation analysis by internal labeling (EMAIL)

“…In addition, certain mismatch endonucleases demonstrate great sensitivity for low concentrations of substrate which is of particular relevance for the detection of rare alleles, since the capacity to pool samples is an important consideration for increasing the throughput of mutation scans [6]. Similarly, for diagnostic applications, high sensitivity increases the capacity to detect disease mutations amongst a pool of contaminating stromal alleles [7].…”

“…A standard adaptation of this approach involves the PCR amplification of pooled sample alleles using 5 0 -labeled oligonucleotide primers and the detection of digestion products by LIF electrophoresis [7,10]. A significant shortcoming of this method, however, is the limited ability to detect signal.…”

Use of Denaturing HPLC for Detection of Mutations in the BCR-ABL Kinase Domain in Patients Resistant to Imatinib

“…In our study, mutations were detected in only a minority of PCR clones in five of the nine patients studied. Other, more recently described mutational screening methods, with sensitivity superior to that of sequencing, include an endonuclease/ ligase-based technique and an enhanced PCR-restriction fragment length polymorphism analysis method (22,23 ). However, both techniques are multistep and not easily amenable to routine screening of clinical samples.…”