Kahn S M, Jiang W, Culbertson T A, Weinstein I B, Williams G M, Tomita N, Ronai Z
Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Oncogene. 1991 Jun;6(6):1079-83.
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.
我们开发了一种快速且高度灵敏的非放射性方法，用于在存在多达10⁴个野生型密码子12等位基因拷贝的情况下检测突变型密码子12人类c-K-ras等位基因。这种灵敏度是通过采用两阶段程序对突变型K-ras基因序列进行选择性聚合酶链反应（PCR）扩增来实现的。第一阶段需要扩增K-ras突变型和野生型密码子12序列，随后仅对野生型序列进行选择性限制性酶切消化。第二阶段涉及对未消化的扩增片段进行后续扩增，这些片段富含突变型密码子12序列。这些产物经过限制性片段长度多态性分析，以检测密码子12处的点突变。该技术快速、非放射性，并且无需寡核苷酸杂交或DNA测序。这种选择性扩增程序的变体可能在检测异质细胞群体中的特定点突变方面具有前景。
