肽核酸钳制PCR技术在结肠癌K-Ras基因突变检测中的优化

目的优化建立一种敏感、简便、稳定地检测结肠癌患者K-Ras基因突变的方法。方法构建K-Ras基因第二外显子12、13密码子的野生型及突变型质粒，通过优化PCR及肽核酸与特异性引物的浓度关系，达到有效模板浓度低的样品K-Ras基因突变的检测。结果成功构建K-Ras基因第二外显子12、13密码子的野生型和突变型质粒。肽核酸钳制PCR条件优化包括：（1） 最佳复性温度为58℃和60℃；（2） 有效模板浓度为10-6pg／μl；（3） 引物浓度与肽核酸浓度的最佳比例为20∶1。在K-Ras突变型质粒与野生型质粒浓度比为1∶100时即可检测到突变。结论肽核酸钳制PCR技术较传统的测序方法更为敏感，可以应用于有效模板浓度低的样品，为结肠癌个体化治疗前相关基因检测的有效方法。

Optimization of peptide nucleic acid clamp PCR for K-Ras mutation in colon cancer

Objective To develop a sensitive,simple and stable method for K-Ras mutation detection in patients of colon cancer.Methods We built wild-type and mutant-type plasmids of the second exon in K-Ras gene and optimized the mixture of polyamide nucleic acid（PNA） and primers to detect low-level mutations.Results The wild type and mutation type plasmids of 12 and 13 codon of the second exon in K-Ras gene was successfully constructed.The conditional optimization of polyamide nucleic acid clamp PCR（PNA-PCR）included：（1） the best temperature of renaturation was 58℃ and 60℃,（2） the effective template concentration was 10^-6pg/μl,（3） the best ratio of primer and PNA concentration was 20∶1.The mutation of K-Ras gene could be detected when the concentration ratio of mutation type and wild type plasmids was 1∶100.Conclusion PNA-PCR method is more sensitive for samples of low concentration template than direct sequencing,and plays an effective role in gene testing before individualized therapy of colon cancer.