焦磷酸测序法快速检测胰腺癌K-ras基因点突变

目的 建立基于焦磷酸测序技术的胰腺癌K-ras基因点突变的检测方法,并与Sanger测序法作一比较.方法 用焦磷酸测序法(Pyrosequencing)和Sanger测序法(Sanger sequencing)分别在10名正常胰腺组织、49例胰腺癌、11例慢性胰腺炎、18例胰腺良性囊肿、7例胰岛素癌、9例壶腹癌、7例胆管癌及7例十二指肠乳头癌石蜡包埋组织的DNA中检测K-rag基因12密码子点突变.结果 采用上述两种方法在所有正常胰腺组织、慢性胰腺炎、胰腺良性囊肿、胰岛素癌、壶腹癌、胆管癌及十二指肠乳头癌均未见K-ras基因点突变,而采用焦磷酸测序法发现胰腺癌石蜡包埋组织K-ras基因点突变率为71.4%(35/49),显著高于采用Sanger测序法发现胰腺癌石蜡包埋组织K-rag基因点突变率(61.2%,30/49).结论 焦磷酸测序法较Sanger测序法更为敏感,且焦磷酸测序法准确、快速、高通量,适合临床标本的批量测定.

Rapid detection of K-ras gene point mutation in pancreatic adenocarcinoma by Pyrosequencing

Objective To establish a method for detecting K-rag gene point mutation in pancreatic adenocarcinoma based on the pyrosequencing and to compare its performance with that of Sanger sequencing.Methods Genomic DNA was extracted from formalin-fixed,paraffin-embedded pancreatic tissues including 49 pancreatic adenocarcinoma,10 normal pancreas,11 chronic pancreatitis,18 benign pancreatic tumor,7 insulin carcinoma,9 ampullary carcinoma,7 bile duct carcinoma and 7 duodenum papillary adenocarcinoma tissues.K-rag gene point mutations at codon 12 were analyzed by pyrosequencing and Sanger sequencing,respectively.Results No mutant K-ras gene Wag detected in normal pancreas,chronic pancreatitis,benign pancreatic tumor,insulin carcinoma,ampullary carcinoma,bile duct carcinoma and duodenum papillary adenocarcinoma tissues by either pyrosequencing or Sanger sequencing.K-rag gene point mutation was detection rate in pancreatic adenocarcinoma tissues was 71.4%(35/49)by pyrosequencing and 61.2%(30/49)by Sanger sequencing,respectively.Conclusions Pyrosequencing is more sensitive than Sanger sequencing and is also accurate,rapid and of high throushput in detecting mutant K-ras gene.It may serve as a practical method for fast batch analysis of clinical samples.