PCR-SSCP analysis of p16 gene mutation by capillary electrophoresis with laser-induced fluorescence detector
作者：WU Yiming ZHANG Zhenzhong SHI Xianglin
单位：
关键词：p16；gene；gene；mutation；SSCP；high；performance；capillary；electrophoresis；PCR
河南医科大学学报000320WU Yiming,ZHANG Zhenzhong,SHI Xianglin
( Department of Occupational Health and Health Toxicology， Henan Medical University Zhengzhou 450052 P.R.China)(Department of Pharmacy, Henan Medical University, Zhengzhou 450052 P.R.China)(Pathology and Physiology Research, NIOSH, Morgantown, WV 26505, USA)
, 百拇医药
Lung cancer is one of the most common cancers in the world. Some genetic alterations such as p53 gene and ras gene mutations, have been identified in this disease. Recently, a putative tumor suppressor gene, the p16/CDKN2/MTS1 gene containing 3 extrons and 2 introns, located in the chromosome p21 region, was cloned independently by three research groups. Traditionally, gene mutation analysis was performed by slab polyacrylamide gel electrophoresis. However, this method is laborious, time-consuming, low sensitivity and harmful to human health. Capillary electrophoresis (CE) with the characteristics of rapidity and high performance has numerous advantages over conventional slab polyacrylamide gel electrophoresis. An important advantage of CE is that the commercially available system is automation.
, 百拇医药
A fused silica capillary (37 cm×75 μm) was precoated by use of the method of Hjerten with some modifications. DNA was isolated from mouse lung tissues by proteinase K digestion and phenol-chloroform extraction using the method of Sambrook with minor modifications. Primers used in this experiment were as the following: upstream primer, 5'-TTC/CTG/GAC/ACG/CTG/GTG/GT-3'; downstream primer, 5'-TCT/GAG/CTT/TGG/AAG/CTC/TCA/G-3'. All PCR reactions were performed in a DNA cycle for 35 cycles and the length of the generated PCR product was 240 bp. The SSCP analysis was carried out using a P/ACE (Beckman) capillary electrophoresis with laser-induced fluorescent detector (λex=488 nm,λem=513 nm). The capillary was filled with 4% short chain linear polyacrylamide (SLPA) containing 100 mmol/L Tris +100 mmol/L boric acid + 2 mmol/L EDTA + 5% glycerol + 2 μg/L thiazole orange by pressure for 5 min and preconditioned in running buffer for 10 min. After each run the capillary coated was purged with water for 5 min and again filled with 4% SLPA. Prior to electrophoresis, the sample was diluted 6 folds with sample buffer (consisting of 20 mmol/L EDTA and 96% formamide deionized ) and heated at 95 ℃ for 5 min and then immediately chilled in ice water bath for 5 min. After adding of thiazole orange to the above sample solution, injections were performed at reversed polarity of 5 kV for 15 s and the separations were carried out under the constant voltage of 7 kV. The signals were collected by laser-induced fluorescent detector and the data were processed by Gold system software. At the concentration of 4%, 20 normal samples and 16 tumor samples in experiment group(30 cases) were detected and the results revealed that sample 10 (tumor) had one abnormal peak (at about 18 min) compared with normal sample, sample18 (tumor) had two abnormal peaks (at about 17.2 min and 18.2 min). Therefore, CE is very suitable for SSCP analysis by commercially available capillary electrophoresis instrument.
基金项目： 河南省自然科学基金资助项目 204022300
2000-01-05收稿, http://www.100md.com
单位：
关键词：p16；gene；gene；mutation；SSCP；high；performance；capillary；electrophoresis；PCR
河南医科大学学报000320WU Yiming,ZHANG Zhenzhong,SHI Xianglin
( Department of Occupational Health and Health Toxicology， Henan Medical University Zhengzhou 450052 P.R.China)(Department of Pharmacy, Henan Medical University, Zhengzhou 450052 P.R.China)(Pathology and Physiology Research, NIOSH, Morgantown, WV 26505, USA)
, 百拇医药
Lung cancer is one of the most common cancers in the world. Some genetic alterations such as p53 gene and ras gene mutations, have been identified in this disease. Recently, a putative tumor suppressor gene, the p16/CDKN2/MTS1 gene containing 3 extrons and 2 introns, located in the chromosome p21 region, was cloned independently by three research groups. Traditionally, gene mutation analysis was performed by slab polyacrylamide gel electrophoresis. However, this method is laborious, time-consuming, low sensitivity and harmful to human health. Capillary electrophoresis (CE) with the characteristics of rapidity and high performance has numerous advantages over conventional slab polyacrylamide gel electrophoresis. An important advantage of CE is that the commercially available system is automation.
, 百拇医药
A fused silica capillary (37 cm×75 μm) was precoated by use of the method of Hjerten with some modifications. DNA was isolated from mouse lung tissues by proteinase K digestion and phenol-chloroform extraction using the method of Sambrook with minor modifications. Primers used in this experiment were as the following: upstream primer, 5'-TTC/CTG/GAC/ACG/CTG/GTG/GT-3'; downstream primer, 5'-TCT/GAG/CTT/TGG/AAG/CTC/TCA/G-3'. All PCR reactions were performed in a DNA cycle for 35 cycles and the length of the generated PCR product was 240 bp. The SSCP analysis was carried out using a P/ACE (Beckman) capillary electrophoresis with laser-induced fluorescent detector (λex=488 nm,λem=513 nm). The capillary was filled with 4% short chain linear polyacrylamide (SLPA) containing 100 mmol/L Tris +100 mmol/L boric acid + 2 mmol/L EDTA + 5% glycerol + 2 μg/L thiazole orange by pressure for 5 min and preconditioned in running buffer for 10 min. After each run the capillary coated was purged with water for 5 min and again filled with 4% SLPA. Prior to electrophoresis, the sample was diluted 6 folds with sample buffer (consisting of 20 mmol/L EDTA and 96% formamide deionized ) and heated at 95 ℃ for 5 min and then immediately chilled in ice water bath for 5 min. After adding of thiazole orange to the above sample solution, injections were performed at reversed polarity of 5 kV for 15 s and the separations were carried out under the constant voltage of 7 kV. The signals were collected by laser-induced fluorescent detector and the data were processed by Gold system software. At the concentration of 4%, 20 normal samples and 16 tumor samples in experiment group(30 cases) were detected and the results revealed that sample 10 (tumor) had one abnormal peak (at about 18 min) compared with normal sample, sample18 (tumor) had two abnormal peaks (at about 17.2 min and 18.2 min). Therefore, CE is very suitable for SSCP analysis by commercially available capillary electrophoresis instrument.
基金项目： 河南省自然科学基金资助项目 204022300
2000-01-05收稿, http://www.100md.com