Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction.
Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction.
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DOI：
10.1186/1472-6750-3-11
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发表时间：
2003-07-23
影响因子：
3.5
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中科院分区：
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识别细胞群体中的已知突变对于临床应用和基础癌症研究非常重要。在这项工作中，一种被称为Polony技术的固定形式的聚合酶链式反应被用于检测癌细胞系中的突变和基因缺失，从而导致杂合性丢失(LOH)。具体地说，在胰腺癌细胞系PANC-1中，对p53的突变热点即密码子175、245、248、249、273和282以及K-ras2、密码子12、13和61进行了基因分型。此外，还对PANC-1中的这两个基因进行了杂合性缺失分析，量化了P53和K-ras2的相对基因拷贝数。用Polony技术检测到PANC-1只有一个P53拷贝和两个K-ras2拷贝，分别含有密码子273的突变和K-ras2的野生型和12密码子突变。为了进一步说明这种方法的一般方法，我们还用Polony技术检测了胰腺癌细胞株ASPC-1和Capan-1中的K-ras2突变。总之，我们开发了一种检测方法，可以检测P53和K-ras2热点的突变，并诊断这些基因的杂合性丢失。
The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH), in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2. Using polony technology, Panc-1 was determined to possess only one copy of p53, which possessed a mutation in codon 273, and two copies of K-ras2, one wildtype and one with a mutation in codon 12. To further demonstrate the general approach of this method, polonies were also used to detect K-ras2 mutations in the pancreatic cell lines, AsPc-1 and CAPAN-1. In conclusion, we have developed an assay that can detect mutations in hotspots of p53 and K-ras2 as well as diagnose LOH in these same genes.