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PCR循环测序法检测K-ras和HCV 5’非编码区基因变异

DETECTION OF NUCLEOTIDE VARIATIONS OF Kras AND HCV 5′ NTR GENES BY PCRBASED CYCLE DNA SEQUENCING

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【作者】 任秀容欧文晖李全贞周新宇何蕴韶

【Author】 Ren Xiurong Ou Wenhui Li Quanzhen Zhou Xinyu He Yunshao(Institute of Biotechnology, Sun Yatsen University of Medical Sciences, Guangzhou, 510089)

【机构】 中山医科大学生物技术研究所

【摘要】 目的:建立一种简单、快速、可靠的DNA序列分析方法,并利用此方法分析Kras基因和HCV5′非编码区基因的变异情况。方法:PCR循环测序法是将PCR扩增与核酸序列分析相结合的一种研究方法。根据此技术原理,建立了以PCR扩增引物为测序引物,利用TaqDNA聚合酶、荧光标记的2′,3′双脱氧核苷三磷酸(ddNTP)直接进行PCR扩增产物序列分析的方法。应用该方法对人肺癌组织中的Kras癌基因和丙型肝炎病毒5′非编码区(HCV5′NTR)进行了序列测定。结果:肺癌组织中的Kras基因第1外显子第35位碱基发生点突变(GGT→GAT);属于高度保守区的HCV5′NTR存在基因变异。结论:PCR循环测序法具有简单、快速、结果可靠等特点,为基因突变的检测和病原体的核酸序列分析提供了一个快速实用的方法。

【Abstract】 Objective:Establish a simple, rapid and accurate DNA sequence analysis method, utilize this methed to detect the Kras and HCV 5′NTR genes variations. Methods:PCRbased cycle DNA sequencing is a method that combines PCR amplification with DNA sequencing. According to the principle, the PCR amplification fragment was subjected to direct sequencing using Taq DNA polymerase and dyelabeled ddNTP performed on an ABI 310 automated DNA sequencer. The nucleotide variations of Kras gene in the tissue of lung cancer and HCV 5′NTR gene were evaluated by means of the PCRbased cycle DNA squencing. Results:The results showed that the point mutation, GGT→GAT transition, was found in exon 1(base 35) of Kras gene. The nucleotide variations were also identified in HCV 5′NTR gene, which was a highly conserved region of HCV genome. Conclusions:The experiment demonstrated that the PCRbased cycle DNA sequencing posses the characteristics of simplicity, rapidity and accuracy, which provides a rapidly practical method for the detection of nucleotide variations of specific gene and DNA sequence of pathogen.

  • 【文献出处】 中山医科大学学报 ,ACADEMIC JOURNAL OF SUN YAT-SEN UNIVERSITY OF MEDICAL SCIENCES , 编辑部邮箱 ,1998年02期
  • 【分类号】Q319.3-3,
  • 【下载频次】68
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