Ligation of a primer at a mutation: a method to detect low level mutations in DNA
Ligation of a primer at a mutation: a method to detect low level mutations in DNA
复制标题
DOI：
10.1093/mutage/17.5.365
复制
发表时间：
2002-09-01
期刊：
影响因子：
2.7
通讯作者：
Makrigiorgos, GM
中科院分区：
文献类型：
--
作者：
Kaur, M;Zhang, YZ;Makrigiorgos, GM
在暴露于诱变剂后或在癌症发展的早期阶段检测低频突变有助于风险评估和分子诊断。我们提出了一种灵敏的新方法来检测大量过量野生型序列内诱导的痕量DNA突变。该方法是基于突变诱导产生新的限制性内切酶识别位点。使用高保真聚合酶从基因组DNA或cDNA扩增DNA序列。纯化的PCR产物用识别新产生的限制性位点的限制性酶消化，部分去磷酸化并在突变位置与寡核苷酸连接。然后在两轮PCR中利用连接的寡核苷酸扩增突变的DNA，但不扩增不含限制性位点的野生型等位基因。mRNA中的A-->T多态性（腱蛋白基因，A（2366）-->T，Asn-->Ile）和基因组DNA中的G-->A多态性（Ku基因，G（74582）-->A，瓦尔-->Ile），这两者都产生酶SAU 3A 1的限制性位点，证明了该应用。11个预先表征修复基因Ku中G（74582）-->A多态性的患者样品用于证明该方法的可靠性。该技术定量检测Ku G-->A多态性，相对于野生型等位基因的突变频率为1.6 × 10（-6）。使用本方法可以容易地检测经常由诱变剂诱导的p53突变。例如，使用第二种酶BbvI，在患者样品中检测到人类癌症中经常遇到的突变（G（14154）-->A突变，p53密码子245，Arg-->Gln）。该方法不需要放射性，利用已建立的程序，并克服了已知在基于RFLP的测定中产生假阳性的几个因素。本扩增通过引物连接在突变（APRIL-ATM）在检测诱变剂产生的遗传改变，早期检测肿瘤标志物突变的身体排出物和微小残留病的诊断有潜在的应用。
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.