Improved detection of mutations in the p53 gene in human tumors as single-stranded conformation polymorphs and double-stranded heteroduplex DNA.
Improved detection of mutations in the p53 gene in human tumors as single-stranded conformation polymorphs and double-stranded heteroduplex DNA.
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改进了人类肿瘤中 p53 基因突变的检测，作为单链构象多态性和双链异源双链 DNA。
DOI：
10.1101/gr.2.1.96
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Sukumar,S
中科院分区：
文献类型：
--
作者：
Soto,D;Sukumar,S
过去3年的研究已经越来越清楚地表明，许多人类肿瘤含有突变的P53肿瘤抑制基因(参考文献[1])。1)。由于突变通常分布在至少5个外显子(外显子5-9)上，跨越2.9kb的编码序列，已开发出更快的扫描该区域的方法。~2)检测p53基因点突变和缺失的流行方法是单链构象多态(SSCP)。(2‘3)该技术基于携带突变的单链DNA片段的固体支撑而改变的迁移速度。DNA迁移的改变可能是由单链DNA构象的差异引起的。该方法在很大程度上依赖于优化构象多态迁移差异的实验条件。因此，向聚丙烯酰胺中添加甘油、降低温度、增加运行长度等会导致更高水平的重现性。2‘3~尽管有这些改进，但使用聚丙烯酰胺作为凝胶载体时，重复性和分辨率存在反复出现的问题，因此需要新的聚合物来实现这一目的。还需要开发一种简单的非放射性技术，用于在临床实验室中筛查p53基因的突变。一种利用野生型和携带突变的DNA片段迁移率差异的凝胶电泳技术可能被证明对这一目的有用。作为异源双链的双链DNA(HTX)在固体载体中的迁移速度与作为同源双链的DNA的迁移速度不同。(4‘S~通过溴化乙锭染色可以很容易地看到这些差异。然而，聚丙烯酰胺凝胶载体对这两种物种的分辨能力因DNA片段的序列而异。4‘S~为了提高放射性单链构象多态性分析的灵敏度，并拆分非放射性双链体，我们开始了其他聚合物的实验。我们在相同的实验条件下测试了聚丙烯酰胺、HHLINK-D5000和HHLINK-MDE在有或没有10%甘油的情况下。我们发现，在SSCP中检测单链DNA和HTX分析中的DNA双链时，HYLINK-MDE(AT Biochem，Malven，PA)，一种乙烯基聚合物，提高了条带的分辨率和重复性。因为SSCP分析不能检测到不会导致迁移改变的多态的突变，所以SSCP分析和HTX分析相结合可能比单独使用其中任何一种方法提供更可靠的结果。用5%聚丙烯酰胺对人乳腺肿瘤进行单链构象多态性分析，检测P53外显子5-9的突变情况。6)以正常乳腺组织细胞系(HBL-100)为阴性对照，乳腺癌细胞株为阳性对照(SKBR3和HS578外显子5，T47D外显子6，MW外显子7，BT474外显子8)。以结肠癌细胞株SW480为阳性对照，检测p53基因外显子9的突变情况。应该注意的是，这些携带p53基因不同外显子突变的细胞株已经失去了p53基因的正常等位基因，因此是半合子或纯合子突变。对原发乳腺肿瘤的DNA也进行了聚丙烯酰胺的单链构象多态性分析，发现59例原发肿瘤中有10例(17%)在P53基因的这个区域发生了突变。(6)然后，用…的一种改进的基于聚丙烯酰胺的乙烯基聚合物--氢链-MDE对同一组细胞系的DNA以及聚丙烯酰胺凝胶(PGE)上可疑阳性的原发肿瘤的DNA进行了重新检测
Research in the last 3 years has made it increasingly clear that a large variety of human tumors contain mutated p53 tumor suppressor genes (reviewed in ref. 1). Since mutations are most often distributed over at least 5 exons (exons 5-9) spanning 2.9 kb of coding sequence, faster methods for scanning the region have been developed.~ 2) A popular method for detection of point mutations and deletions in the p53 gene is single-stranded conformation polymorphism (SSCP).(2'3) The technique is based on altered migration speeds through solid supports of singlestranded DNA fragments carrying mutations. The altered migration of DNA is presumably caused by differences in the conformation of single-stranded DNA. The method depends heavily on experimental conditions that optimize migration differences of the conformation polymorphs. Thus, adding glycerol to the polyacrylamide, reducing the temperature, increasing the length of the run, and so forth result in a greater level of reproducibility. 2'3~ In spite of these modifications, reproducibility and resolution present recurrent problems using polyacrylamide as a gel support, presenting the need for novel polymers for this purpose. There is also a need to develop a simple nonradioactive technique for screening for mutations in the p53 gene in the clinical laboratory. A gel electrophoresis technique that takes advantage of differences in mobility of wild-type and mutation-bearing DNA fragments could prove useful for this purpose. Double-stranded DNA as heteroduplexes (HTX) migrate at different rates compared to DNA as homoduplexes in solid supports.(4's~ These differences can be visualized easily by staining with ethidium bromide. However, the resolution ability of polyacrylamide gel supports for detection of the two species varies with the sequence of the DNA fragments. 4's~ In an effort to improve the sensitivity levels of the radioactive SSCP analysis, and to resolve the nonradioactive duplexes, we began to experiment with other polymers. We tested polyacrylamide, Hydrolink-D5000, and Hydrolink-MDE under identical experimental conditions with and without 10% glycerol. We found that Hydrolink-MDE (AT Biochem, Malvern, PA), a vinyl polymer, gave improved resolution of bands as well as reproducibility in the detection of both single-stranded DNA in SSCP and of DNA duplexes in the HTX analyses. Because the SSCP analysis is unable to detect mutations that do not result in polymorphs with altered migrations, a combination of SSCP analysis with HTX analysis might provide more reliable results than either one alone. Human mammary tumors were analyzed by SSCP for mutations in p53 exons 5-9 using 5% polyacrylamide. 6) A cell line derived from normal breast tissue (HBL-100) was used as a negative control, whereas breast tumor cell lines served as positive controls (SKBR3 and HS578 for exon 5, T47D for exon 6, MW for exon 7, and BT474 for exon 8). The colon cancer cell line, SW480 was used as a positive control for mutation in exon 9 of the p53 gene. It should be noted that these cell lines, carrying mutations in the various exons of the p53 gene, have lost the normal allele of the p53 gene and are therefore, hemi-or homozygous mutants. Primary breast tumor DNAs were also analyzed by SSCP on polyacrylamide and it was found that 10 out of 59 (17%) primary tumors had mutations in this region of the p53 locus.(6) Then, DNA from the same panel of cell lines as well as primary tumors considered to be questionable positives by SSCP analysis on polyacrylamide gels (PGE) was retested with the polymer, Hydrolink-MDE (MDE), a modified polyacrylamide-based vinyl polymer …