杂合突变的PCR产物中包含异源双链DNA片段的研究

目的:验证杂合突变的PCR产物中包含异源双链DNA片段.方法:采用PCR、PAGE电泳和变性高效液相色谱分析的方法,对中国人表皮松解性掌跖角化症致病基因KRT9第1外显子的杂合的碱基插入/缺失突变(497delAinsGGCT)进行实验验证.结果:KRT9基因第1外显子碱基插入/缺失突变(497delAinsGGCT)杂合子的PCR产物,本身包含2种异源双链DNA片段和2种同源双链DNA片段,不需要经过额外的变性、退火处理.结论:在用DGGE、TGGE、DHPLC和HA等需要形成杂合双链体的基因突变检测技术进行突变检测时,如果突变为杂合子,则PCR产物可以直接进行突变检测,不需要预先加热/冷却等预处理以形成异源双链DNA分子.

PCR products with heterozygous mutations containing two types of heteroduplexes

Objective: To confirm that PCR products with heterozygous mutations contain not only wide-type and mutant homoduplexes,but also two types of heteroduplexes.Methods: An insertion-deletion mutation in the exon 1 of KRT9 gene(497delAinsGGCT),which caused Chinese epidermolytic palmoplantar keratoderma(EPPK) was investigated by polymerase chain reaction(PCR),polyacrylamide gel electrophoresis(PAGE) and denaturing high-performance liquid chromatography(DHPLC).Results: Two heteroduplexes and two homoduplexes in the PCR product from the heterozygous mutation of the exon 1 of KRT9(497delAinsGGCT) were detected.Conclusion: PCR products from KRT9 gene with heterozygous mutations contain two types of heteroduplexes.It is without the need to perform heating and cooling PCR products obtained from heterozygous mutations in advance before the mutation screening steps such as denaturing gradient gel electrophoresis(DGGE),temperature gradient gel electrophoresis(TGGE),conformation-sensitive gel electrophoresis(CSGE),DHPLC and heteroduplex analysis(HA),etc.