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

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。

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.