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Genetic Analysis of Paraffin-Embedded Human Tumor Samples by GC-clamped DGGE.

通过 GC 钳位 DGGE 对石蜡包埋的人类肿瘤样本进行遗传分析。

基本信息

批准号：
04557018
负责人：
OGAWA Katsuhiro
金额：
$ 1.79万
依托单位：
Asahikawa Madical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04557018/
关键词：
PCR GC clamp DGGE ras gene p53 mutations paraffin-embedded tissues human tumors point mutation

项目摘要

We established the GC-clamped DGGE method which can detect genetic alterations in small pieces of tissue samples without use of radioisotopes. This method was applicable to analysis of human tumors enucleated from tissue sections. In practice, GC-rich sequences were introduced to one side of PCR fragments using GC-clamped PCR primers. When these PCR products were electrophoresis on gels which vertically contained a 0-80% gradient of denaturing agents (urea and formamide), electrophoretic mobility was retarded over a certain concentration of the denaturants at which conformational changes of DNA occurred. When the PCR products contained mutated sequences, electrophoresis patterns were differed to that of the wild type cases. Many samples can be simultaneously analyzed by electrophoresing the samples to the parallele direction of gradient.It was revealed that the method was sensitive enough to detect mutations in one part of mutated DNA diluted with 16 parts of wild type DNA.Following results were obtained using this method.1)K-ras codon 12 mutations were highly frequent in human bile duct and gallbladder carcinomas. These mutations were mainly a glycine to serine change.2)K-ras and p53 mutations were compared between mucin-producing cystic type and ordinary form of human pancreatic cancers. Although K-ras codon 12 mutations were detected in most cases of both types, p53 mutations were found only in the ordinary form.3)p53 mutations were highly frequent in rat hepatocellular carcinoma cell (HCC) lines. However, such mutations were rarely observed in chemically-induced rat HCCs, indicating that the p53 mutations may occur during establishment of HCC cell lines.4)K-ras condon 12 mutations were detected at low frequency in rat HCCs. However, frequency of the mutations was different depending on HCC-inducing agents.5)p53 mutations were rarely detected in spontaneously-immortalized mouse embryonic fibroblasts and their transformed derivatives.

我们建立了气相色谱夹固DGGE方法，该方法可以在不使用放射性同位素的情况下检测小块组织样本的遗传改变。该方法适用于人组织切片去核肿瘤的分析。在实际操作中，利用gc夹紧的PCR引物将富含gc的序列引入到PCR片段的一侧。当这些PCR产物在垂直含有0-80%梯度的变性剂（尿素和甲酰胺）的凝胶上电泳时，在一定浓度的变性剂发生DNA构象变化时，电泳迁移速度减慢。当PCR产物含有突变序列时，电泳模式与野生型病例不同。将样品沿平行梯度方向电泳，可同时分析多个样品。结果表明，该方法足够灵敏，可以在16份野生型DNA稀释的突变DNA中检测到一部分突变。用这种方法得到了以下结果：1)K-ras密码子12突变在人胆管癌和胆囊癌中非常常见。这些突变主要是甘氨酸到丝氨酸的变化。2)比较产粘蛋白囊型和普通型人胰腺癌中K-ras和p53的突变。虽然K-ras密码子12突变在两种类型的大多数病例中都检测到，但p53突变仅在普通形式中发现。3)p53突变在大鼠肝细胞癌（HCC）细胞系中非常常见。然而，在化学诱导的大鼠HCC中很少观察到这种突变，这表明p53突变可能发生在HCC细胞系的建立过程中。4)在大鼠hcc中低频率检测到K-ras安全套12突变。然而，突变的频率因hcc诱导剂的不同而不同。5)在自发永生化的小鼠胚胎成纤维细胞及其转化衍生物中很少检测到p53突变。