Ligation of a primer at a mutation: a method to detect low level mutations in DNA

Abstract

Detection of low frequency mutations following exposure to mutagens or during the early stages of cancer development is instrumental for risk assessment and molecular diagnosis. We present a sensitive new method to detect trace levels of DNA mutations induced within a large excess of wild-type sequences. The method is based on mutation-induced generation of new restriction enzyme recognition sites. A DNA sequence is amplified from genomic DNA or cDNA using a high fidelity polymerase. The purified PCR product is digested with a restriction enzyme that recognizes the newly generated restriction site, partially dephosphorylated and ligated with an oligonucleotide at the position of the mutation. The ligated oligonucleotide is then utilized in two rounds of PCR to amplify the mutated DNA but not the wild-type allele that contains no restriction site. An A-->T polymorphism in mRNA (tenascin gene, A(2366)-->T, Asn-->Ile) and a G-->A polymorphism in genomic DNA (Ku gene, G(74582)-->A, Val-->Ile), both of which generate a restriction site for the enzyme SAU3A1, demonstrate the application. Eleven patient samples pre-characterized for the G(74582)-->A polymorphism in the repair gene Ku are used to demonstrate the reliability of this approach. This technique quantitatively detects the Ku G-->A polymorphism at a mutant frequency of 1.6x10(-6) relative to the wild-type allele. Mutations in p53 that are frequently induced by mutagens can readily be detected using the present method. As an example, using a second enzyme BbvI, a mutation frequently encountered in human cancers (G(14154)-->A mutation, p53 codon 245, Arg-->Gln) was detected in patient samples. The process does not require radioactivity, utilizes established procedures and overcomes several factors known to produce false positives in RFLP-based assays. The present amplification via primer ligation at the mutation (APRIL-ATM) has potential applications in the detection of mutagen-generated genetic alterations, early detection of tumor marker mutations in bodily discharges and the diagnosis of minimal residual disease.

PAP: Detection of ultra rare mutations depends on P* oligonucleotides: ?Sleeping Beauties? awakened by the kiss of pyrophosphorolysis

“…RFLP/PCR, the most sensitive of the methods, has been reported to detect a mutated allele in 10 8 wild-type alleles [Felley-Bosco et al, 1991], but 1) the method is highly labor intensive, 2) the mutation must be within an appropriate restriction endonuclease site, and 3) the cleavage efficiencies of many restriction endonucleases limit the selectivity of the method to a range of 1:10 3 to 1:10 5 [Parsons and Heflich, 1997b]. PCR/RFLP and its many variants, such as quantitative enriched PCR [Ronai and Minamoto, 1997], inverse restriction site mutation analysis [Jenkins et al, 1999], modified nucleotide analogs and buffers [Day et al, 1999], and modification using ligation [Kaur et al, 2002], achieved a selectivity of 1:10 6 [van Mansfeld and Bos, 1992]. ACB-PCR detected a mutated allele in 10 5 wild-type alleles [Parsons and Heflich, 1998a] and its combination with MutEX enrichment increased the selectivity to 1:10 7 [Parsons and Heflich, 1998b].…”

Inverse PCR-Based RFLP Scanning Identifies Low-Level Mutation Signatures in Colon Cells and Tumors

“…Independent verification that iFLP-generated DNA amplicons correspond to genuine mutations at the corresponding circularized fragment was obtained by PCR amplifying directly from genomic DNA the indicated DNA region, followed by amplification via primer ligation at the mutation (23,24). This approach uses the formation of a TaqI site at the mutation to ligate a linker and selectively PCR-amplify mutation-containing alleles.…”

Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens

“…WTB-PCR is no more complex or time consuming than standard PCR and requires no additional apparatus. Any approach that uses PCR amplification followed by analysis of the amplified DNA for the presence of mutations will have its sensitivity limited by the frequency of PCR errors (Parsons and Heflich, 1997; Kaur et al, 2002;Liu et al, 2002). A fundamental advantage of WTB-PCR is that the blocker oligonucleotide prevents amplification of wild-type DNA thereby preventing amplification of any template not containing mutations in the targeted region of the gene.…”