Mutation detection using a novel plant endonuclease
Mutation detection using a novel plant endonuclease
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DOI：
10.1093/nar/26.20.4597
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发表时间：
1998-10-15
影响因子：
14.9
通讯作者：
Yeung, AT
中科院分区：
文献类型：
--
作者：
Oleykowski, CA;Mullins, CRB;Yeung, AT
我们发现了一种用于突变检测的新试剂--芹菜中的一种新的核酸酶CEL I。它专用于从pH 6到9的DNA扭曲和错配，切割是在异源双链中两条DNA链之一的错配位置的3‘侧。Cel I类核酸酶存在于许多植物中。我们在这里报告了一种使用CEL I的简单的酶突变检测方法，可以有效地识别突变和多态。为了验证这种方法的有效性，用两种颜色的荧光染料标记的5‘标记的引物进行了BRCA1基因外显子的聚合酶链式反应，扩增产物经退火形成异源双链，再经CEL I酶切。在使用PE应用生物系统自动DNA测序仪进行的GeneScan分析中，两个独立的切割事件，每条链上一个，产生两种颜色的截断片段，相互补充，以确定错配的位置。CEL I可以检测100%的序列变异，包括缺失、插入和错义改变，我们的结果表明CEL I突变检测是一种高度敏感的方法，可以检测长度长达1120bp的DNA片段的多态和致病突变。
We have discovered a useful new reagent for mutation detection, a novel nuclease CEL I from celery. It is specific for DNA distortions and mismatches from pH 6 to 9, Incision is on the 3'-side of the mismatch site in one of the two DNA strands in a heteroduplex. CEL I-like nucleases are found in many plants. We report here that a simple method of enzyme mutation detection using CEL I can efficiently identify mutations and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors, The PCR products were annealed to form heteroduplexes and subjected to CEL I incision. In GeneScan analyses with a PE Applied Biosystems automated DNA sequencer, two independent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of the mismatch. CEL I can detect 100% of the sequence variants present, including deletions, insertions and missense alterations, Our results indicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease-causing mutations in DNA fragments as long as 1120 bp in length.