Denaturing high pressure liquid chromatography (DHPLC) for the analysis of somatic p53 mutations
Denaturing high pressure liquid chromatography (DHPLC) for the analysis of somatic p53 mutations
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DOI：
10.1038/labinvest.3780387
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发表时间：
2001-12-01
影响因子：
5
通讯作者：
Höfler, H
中科院分区：
文献类型：
--
作者：
Keller, G;Hartmann, A;Höfler, H
变性高压液相色谱(DHPLC)是一种相对较新的技术，它利用野生型和突变DNA链之间的异源双链形成来识别突变。通过离子对，反相液相色谱在一个特殊的柱基质上部分热变性DNA链来分离异双链分子(O‘Donovan等人，1998；Oefner等人，1998)。对于大量样本的突变筛选，DHPLC可能是一种非常有用的方法。到目前为止，它主要用于分析已报道高度敏感的各种遗传病的种系突变(Gross等人，1999；Holinski-Feder等人，2001；O‘Donovan等人，1998)。然而，在肿瘤的体细胞突变分析方面，一个潜在的缺陷是DHPLC要求同时存在野生型和突变型DNA，以便形成异源双链。理想情况下，这些DNA分子应该以等量存在。在实际的肿瘤标本中，情况往往并非如此，因为在具有抑癌基因体细胞突变的肿瘤中，非肿瘤细胞可能以不同的数量存在，或者正常的野生型等位基因可能丢失(LOH)。在这里，我们报告了使用DHPLC技术来分析体细胞P53突变。我们建立了已知的p53突变外显子5至8的分离条件，用野生型和突变型PCR产物稀释系列测定了检测异源双链的敏感性，然后分析了18例显微解剖的结直肠癌组织中p53外显子5至8的突变。这些肿瘤标本的DNA来自冰冻或石蜡包埋的肿瘤组织，几乎全部由触摸制备(Saitoh et al，1994)或激光显微切割获得的肿瘤细胞组成。用标准技术提取DNA。为建立分离条件，将肿瘤标本和细胞系与野生型DNA按2：1比例混合后进行DHPL分析。扩增条件为25 L，反应混合液为10 mM Tris-HCl(pH 8.3)，50 mM KCl1，1.0，1.5或2.0 mM氯化镁，0.01%明胶，200 mM dNTP，每个引物0.4 mM。在94℃下进行4分钟的初始变性步骤后，执行40个循环，包括在55~60℃下30秒和在72℃下30秒，然后在72℃下最后延长7分钟。引物是：外显子5向前：atgtgtcacttggcctg；外显子5反向：aaccagccctgtctctc；外显子6向前：agggtccccccccccccccc；外显子6反向：cacccttaacccctcctcccc；外显子7向前：caggctcggcggcctcagcagctctcagcg；外显子8向前：ctctccctctctctctctctcg。突变分析基本上是根据Oefner和UnderHill(1998)的方法在自动DHPLC分析系统(Transgenomy，Omaha，Nebraska)上进行的。扩增产物在94℃变性4min，以1C/min的速度冷却至室温，将3~15个L扩增产物加入预热反相柱(DNA-SEP；转基因)。DNA的洗脱在缓冲液A和B的线性乙腈梯度中进行。缓冲液A由0.1M的三乙基乙酸铵(TEAA)和0.1M的TEAA和25%的乙腈组成的缓冲液B组成。…最佳拆分的温度
Denaturing high pressure liquid chromatography (DHPLC) is a relatively new technique, which uses heteroduplex formation between wild-type and mutated DNA strands to identify mutations. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands (O ‘Donovan et al, 1998; Oefner et al, 1998). DHPLC is potentially a very useful method for the screening of a large number of samples for mutations. So far, it has mainly been used for the analysis of germline mutations in various inherited diseases for which a high degree of sensitivity has been reported (Gross et al, 1999; Holinski-Feder et al, 2001; O ‘Donovan et al, 1998). With respect to the analysis of somatic mutations in tumors, however, one potential drawback is that DHPLC requires that both wild-type and mutant DNA are present so that heteroduplexes can form. Ideally, these DNA molecules should be present in equal amounts. In actual tumor specimens this is often not the case, because non tumorous cells may be present in various amounts or the normal wild-type allele may be lost (LOH) in tumors with somatic mutations of tumor suppressor genes. Here we report the use of the DHPLC technique for the analysis of somatic p53 mutations. We established the separation conditions for exons 5 thru 8 with known p53 mutations, determined the sensitivity for the detection of heteroduplex by dilution series of wild-type and mutant PCR products, and then analyzed 18 microdissected colorectal carcinomas for p53 mutations in exons 5 thru 8. DNA from specimens with known mutations in exons 5 thru 8 of p53 included seven breast carcinomas, two urothelial carcinomas, a DNA sample from a Li-Fraumeni patient and two cell lines (HT29 and MDA-MB-435S), and DNA from blood of an individual with a polymorphism in exon 6. DNA from these tumor samples were either from frozen or paraffin-embedded tumor tissue and consisted almost entirely of tumor cells obtained by touch preparation (Saitoh et al, 1994) or laser microdissection. The DNA was isolated using standard techniques. To establish the separation conditions, DHPLC analysis was performed after mixing the tumor samples and cell lines with wild-type DNA at a ratio of 2: 1. The PCR reactions were performed in 25 l of a reaction mixture consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.0, 1.5, or 2.0 mM MgCl2, 0.01% gelatin, and 200 mM dNTP 0.4 mM of each primer. After an initial denaturation step at 94 C for 4 minutes, 40 cycles were performed consisting of 30 seconds at 55 to 60 C and 30 seconds at 72 C, followed by a final extension of 7 minutes at 72 C. The primers were: exon 5 forward: atgtgttcacttgtgccctg; exon 5 revers: aaccagccctgtcgtctctc; exon 6 forward: agggtccccaggcctctgat; exon 6 revers: cacccttaacccctcctccc; exon 7 forward: ccaaggcgcactggcctcatc, exon 7 revers: cagaggctggggcacagcagg; exon 8 forward: ttccttactgcctcttgctt; exon 8 revers: tgtcctgcttgcttacctcg. Mutation analysis was essentially performed according to the method of Oefner and Underhill (1998) on an automated DHPLC analysis system (Transgenomic, Omaha, Nebraska). The PCR products were denatured for 4 minutes at 94 C and cooled to room temperature at a rate of 1 C/minute, and 3 to 15 l of PCR products were applied to a preheated reverse phase column (DNA-Sep; Transgenomic). Elution of the DNA was performed in a linear acetonitrile gradient of buffers A and B. Buffer A consisted of 0.1 M triethylammonium acetate (TEAA) and buffer B of 0.1 M TEAA and 25% acetonitrile. The temperature for optimal resolution of …