Rapid and sensitive nonradioactive detection of mutant K-ras genes via 'enriched' PCR amplification.
Rapid and sensitive nonradioactive detection of mutant K-ras genes via 'enriched' PCR amplification.
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发表时间：
1991-06
期刊：
影响因子：
8
通讯作者：
S. Kahn;Wei Jiang;Culbertson Ta;Weinstein Ib;Williams Gm;N. Tomita;Z. Ronai
中科院分区：
文献类型：
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作者：
S. Kahn;Wei Jiang;Culbertson Ta;Weinstein Ib;Williams Gm;N. Tomita;Z. Ronai
我们开发了一种快速、高灵敏度的非放射性方法，用于检测存在多达10(4)个野生型密码子12等位基因的突变型人类c-K-ras等位基因。这种敏感性是通过采用两阶段程序对突变K-ras基因序列进行选择性聚合酶链反应（PCR）扩增实现的。第一阶段需要扩增K-ras突变体和野生型密码子12序列，然后仅对野生型序列进行选择性限制性内切酶切。第二阶段包括随后扩增未消化的扩增片段，扩增片段富含突变密码子12序列。对这些产物进行限制性长度多态性分析，以检测密码子12的点突变。该技术快速，无放射性，并且不需要进行寡核苷酸杂交或DNA测序。这种选择性扩增程序的变化可能证明有希望检测异种细胞群体中的特定点突变。
We have developed a rapid and highly sensitive nonradioactive method for the detection of a mutant codon 12 human c-K-ras allele in the presence of as many as 10(4) copies of the wild type codon 12 allele. This sensitivity is achieved by selective polymerase chain reaction (PCR) amplification of mutant K-ras gene sequences employing a two stage procedure. The first stage entails the amplification of both K-ras mutant and wild type codon 12 sequences, followed by a selective restriction enzyme digestion of only wild type sequences. The second stage involves a subsequent amplification of undigested amplified fragments, enriched in mutant codon 12 sequences. These products are subject to restriction length polymorphism analysis for the detection of point mutations at codon 12. This technique is rapid, nonradioactive, and eliminates the need for either oligonucleotide hybridization or DNA sequencing. Variations of this selective amplification procedure may prove promising for the detection of specific point mutations in heterogenous cell populations.