焦磷酸测序法快速检测胰腺癌K-ras基因点突变

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

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-ras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-ras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。

Objective To establish a method for detecting K-ras 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-ras gene point mutations at codon 12 were analyzed by pyrosequencing and Sanger sequencing, respectively. Results No mutant K-ras gene was 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-ras 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 throughput in detecting mutant K-ras gene. It may serve as a practical method for fast batch analysis of clinical samples.