A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.
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机译:用于检测低水平突变的高灵敏度方法正在开发中。进行PCR反应,其中通过改变特异性碱基将限制位点引入野生型DNA中。通过同源限制性核酸内切酶(RE)消化野生型DNA会丰富识别位点内具有突变的产物。重新扩增后,通过连接检测反应(LDR)鉴定突变。该PCR / RE / LDR分析最初用于检测已知野生型样品中的PCR错误。在低| Deltap K a |下测量PCR错误。含有Tricine,EPPS和柠檬酸盐的缓冲液,以及含有Tris的相同缓冲液。使用p53抑癌基因248位密码子的Msp I位点上的完美匹配引物,对PCR条件进行了优化,以最大程度地减少PCR错误。但是,由于突变并不总是在预先存在的限制位点内发生,因此采用了通用的PCR / RE / LDR方法需要引入新的限制位点。原则上,使用错配引物的PCR可以改变序列中的特定碱基并产生新的限制性酶切位点。然而,从3'错配引物延伸可能会产生错延伸产物。我们测试了Msp I(CCGG)网站到Taq I网站(TCGA)的转换。使用天然的碱基T错配引物对不能成功进行转化。当在3个末端使用包含6 H,8 H -3,4-二氢嘧啶[4,5- c] [1,2]恶嗪-7-一(Q6)碱基的修饰引物时,转化成功在使用3'天然碱基T引物进行转化PCR之前进行预转化PCR的检测。嘧啶类似物Q6访问T样和C样互变异构体的能力似乎极大地促进了转化。
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