Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations
Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations
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DOI：
10.1093/nar/26.6.1396
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发表时间：
1998-03-15
影响因子：
14.9
通讯作者：
James, CD
中科院分区：
文献类型：
--
作者：
Liu, WG;Smith, DI;James, CD
变性高效液相色谱法（DHPLC）最近被描述为用于筛查DNA样品的单核苷酸多态性和遗传突变的方法。38个DNA，其中22个是先前表征的重排转化基因（RET）或囊性纤维化跨膜传导调节基因（CFTR）突变或多态性的杂合DNA，91%（20/22）的RET或CFTR杂合型标本PCR扩增产物的洗脱图谱与相应的纯合型正常图谱不同;含有杂合RET序列的两个扩增子的谱是否与纯合病例不同是不确定的。为了研究该方法用于检测肿瘤DNA中突变的有用性，在63个恶性神经胶质瘤中检测了在染色体10基因（PTEN）外显子上缺失的每个磷酸酶和张力蛋白同源物的突变，来自这一系列脑肿瘤的17个PTEN PCR产物显示出指示样品杂合性的洗脱曲线，并且在每种情况下，常规测序证实了突变的存在。对63个肿瘤DNA中的每一个的含有外显子1、3和5的PTEN扩增子进行测序，以确定是否有任何突变可能逃脱DHPLC检测，DHPLC共鉴定出40个PCR产物中的37个（92.5%）含有确定的序列变异，而在196个含有纯合正常序列的扩增子中没有发现变异。
Denaturing high performance liquid chromatography (DHPLC) has been described recently as a method for screening DNA samples for single nucleotide polymorphisms and inherited mutations, Thirty-eight DNAs, 22 of which were heterozygous for previously characterized rearranged transforming gene (RET) or cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations or polymorphisms, were examined using DHPLC analysis to assess the accuracy of this scanning method, Ninety-one per cent (20/22) of the PCR amplicons from specimens with heterozygous RET or CFTR sequence showed elution profiles distinct from corresponding homozygous normal patterns; whether the profiles for two amplicons containing heterozygous RET sequence were distinct from homozygous cases was equivocal, To investigate the usefulness of this method for detecting mutations in tumor DNAs, each of the phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) exons were examined for mutations in 63 malignant gliomas, Seventeen PTEN PCR products from this series of brain tumors showed elution profiles indicating sample heterozygosity and in each instance conventional sequencing confirmed the presence of a mutation, PTEN amplicons containing exons 1, 3 and 5 were sequenced for each of the 63 tumor DNAs to determine whether any mutations may have escaped DHPLC detection, and this analysis identified one such alteration in addition to the eight mutations that DHPLC had revealed, In total, DHPLC identified 37 of 40 (92.5%) PCR products containing defined sequence variation and no alterations were indicated among 196 amplicons containing homozygous normal sequence.