单链构象多态性分析K-ras和p16 基因突变
作者：吴逸明 张振中 史香林 王新朝
单位：吴逸明(河南医科大学劳动卫生学与卫生毒理学教研室 郑州 450052)；张振中(河南医科大学药学系 郑州 450052)；史香林(美国职业安全卫生研究所病理生理学研究室 摩根墩 WV 26505 USA)；王新朝(河南医科大学劳动卫生学与卫生毒理学教研室 郑州 450052)
关键词：基因；突变；SSCP；K-ras；p16
河南医科大学学报000304 摘要 目的：研究大鼠肺癌组织K-ras和p16 基因突变情况。 方法： 运用聚合酶链反应-单链构象多态性 (PCR-SSCP) 技术分析肺癌组织K-ras和p16 基因突变。结果：未发现正常大鼠肺组织中K-ras和p16 基因突变, 在肿瘤组织中 K-ras和p16均有2例突变发生。结论： K-ras和p16 基因突变与肺癌发生存在一定的相关性.
, http://www.100md.com
分类号 R734.2
Mutation analysis of K-ras and p16 genes in rat lung tissues by SSCP
WU Yiming,WANG Xinchao
(Department of Occupational Health and Health Toxicology, Henan Medical University， Zhengzhou 450052，P.R.China)
ZHANG Zhenzhong,(Department of Pharmacy, Henan Medical University ，Zhengzhou 450052 P.R.China)
SHI Xianglin，(Pathology and Physiology Research, NIOSH, Morgantown, WV 26505, USA)
, 百拇医药
Abstract Aim: To study mutation of K-ras and p16 genes in rat lung tumor tissues. Method: Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to detect the mutation of K-ras and p16 genes in rat lung tissues. Result: For K-ras and p16 gene mutations were not found in normal group and were observed in 2 out of 16 lung tumor tissues. Conclusion: K-ras and p16 genes were correlative to the incidence of lung tumor to some extent.
, http://www.100md.com
Key words gene; mutation; SSCP; K-ras; p16
Epidemiology study has shown that lung cancer is the second commonest cancer and its incidence is increasing dramatically in recent years. It is of great importance to study the mechanism and to find the early biomarkers for lung cancer. Mutations in cellular ras gene have been strongly implicated in various stages of mammalian tumorigenesis. In human tumors, the mutations identified have largely been localized to codon 12,13 or 61 of three ras genes[1,2] : H-ras, K-ras and N-ras. 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[3～5]. Traditionally, mutations of genes were detected by hybridization with DNA probe, allele-specific PCR and restriction fragment length polymorphism (RFLP) and single strand conformational polymorphism (SSCP). SSCP analysis in these PCR-based methods is the simplest and most sensitive. Previous studies showed that gene mutations, such as base substitution, point mutation, deletion and insertion, could lead to conformational change of single-stranded DNA, so the mobility shifts could be observed on the electrophorogram of non-denatured polyacrylamide gel[6,7].
, 百拇医药
1 Materials and methods
1.1 Materials acrylamide, N, N, N', N'-tetramethylenediamine(TEMED) and ammonium peroxydisulfate(APS) were purchased from Sigma Chemical Co. 2-amino-2-(hydroxymethyl)-1,3-propanediol(Tris), boric acid, PCR Markers, Taq DNA polymerase, dimethyl sulfoxide(DMSO), bluephenol, xylene cyanole FF and formamide deionized were obtained from Sino-American Biotechnology Company(SABC). dNTP was from Promega. Primers were synthesized by CyberSyn Biotechnology Corporation (American).
, http://www.100md.com
1.2 Amplification of K-ras and p16 genes by PCR DNA was isolated from normal and tumor lung tissues of rat, by proteinase K digestion and phenol-chloroform extraction using the method of Sambrook[8]. Primers of K-ras gene used were as the following: upstream primer, 5'-ACA/TGT/TCT/AAT/ATA/GTC/AC-3'; downstream prime, 5'-AAA/GAA/TGG/TCC/TGC/ACC/AG-3'. The PCR was performed in a 50 μl buffer containing 50 pmol of each primer, 100 pmol of dATP, dCTP, dGTP, dTTP, 5 μl of 10×PCR buffer, 0.5 μg template. Prior to PCR, the mixture was predenatured at 95 ℃ for 5 min and then 2.0 u polymerase was added to the above solution. All PCR reactions were performed in a DNA cycle of SABC and the reaction mixture was covered by 30 μl wax. The reaction mixture was subject to 30 cycles at 94 ℃ for 60 s, 60 ℃ for 56 s and 72 ℃ for 33 s, the extension time for the last cycle was 5 min at 72 ℃, the length of DNA fragment generated by PCR was 212 bp. Primers of p16 gene used 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'. The PCR reaction constituents were the same as K-ras gene PCR mixture. The reaction mixture was subject to 35 cycles at 94 ℃ for 60 s, 64 ℃ for 50 s and 72 ℃ for 53 s, the extension time for the last cycle was 5 min at 72 ℃, the length of DNA fragment generated by PCR was 240 bp.
, http://www.100md.com
1.3 SSCP by non-denatured vertical slab PAGE 10 μl of PCR products ( K-ras or p16 gene) was added to equal volume of sample buffer containing 20 mmol/L EDTA, 96% formamide deionized, 0.05% bromphenol blue and 0.05% xylene cyanole FF.The above solution was heated at 97 ℃ for 5 min and then immediately chilled in ice water bath for 5 min. 10 μl of the above solution was applied to each lane of non-denatured polyacrylamide gel which crosslinked with N, N'-methylenebisacrylamide(bis) was used at composition of 6%T, 2%C and 5% glycerol for p16 gene. and at composition of 5%T, 1.2% C and 5% glycerol for K-ras gene. Electrophoresis was performed at 200 V for 4 h at 4 ℃ and the gels were stained with silver.
, http://www.100md.com
2 Results and discussion
The amplification of K-ras and p16 genes by PCR generated a DNA fragment of 212 bp and 240 bp, respectively. The PCR products length was identified by comparing with PCR Markers on a conventional 2% agarose gel (data not shown). The amplification of K-ras gene by PCR generated a DNA fragment of 212 bp in the extron 1. Activation of K-ras oncogene is related to the pathogenesis of various human tumors. The incidence of ras gene mutation in the lung tumor is about 20%. Point mutations which led to ras activation were confined to the codon 12, 13 and 61 of the gene. Point mutations in codon 12 are commonly seen. In the present study, the PAGE-SSCP results showed that: no mobility shift appeared in the electrophorogram for the 20 normal tissues, but mobility shift appeared for the samples 10 and 12 in the experiment group (30 cases) in which tumor took place in 16 cases. The migration patterns of K-ras gene from normal lung tissues and lung tumor tissues were shown in Fig 1. There were abnormal band in the lane 1 ( sample 10) and lane 3 ( sample 12 ) in the electrophorogram. This result demonstrated that K-ras gene of samples 10 and 12 mutated in the extron 1.
, 百拇医药
p16 gene (multiple tumor suppression gene) contains3 extrons, which contains 126 bp in the extron 1, 307 bp in the extron 2 and 11 bp in the extron 3 respectively and frequently mutates in extron 2. The vertical slab PAGE-SSCP results showed that: no mobility shift was observed in 20 normal tissues, but mobility shifts were observed in 2 out of 16 tumor samples. The migration patterns of p16 gene from normal lung tissues and tumor lung tissues were shown in Fig 2. In the tumor samples, an abnormal band of allelic single-stranded DNA molecular appeared (lane 2 (sample 10) and lane 7 ( sample 18)) in the electrophorogram. The result demonstrated that p16 gene mutated in samples 10 and 18.
, http://www.100md.com
Fig1 Electrophorogram of SSCP for K-ras gene
N: Normal, M: Mutation
Fig2 Electrophorogram of SSCP for p16 gene
N: Normal, M: Mutation
基金项目：河南省自然科学基金资助项目 204022300
作者简介：吴逸明：男，55岁，教授，博士生导师，研究方向：肺癌的病因学、预防、早期诊断和综合治疗的研究，References
[1]Glenna CB, Peter SR, Lawrence AL. Analysis of c-ki-ras mutations in human colon carcinoma by cell sorting, polymerase chain reaction, and DNA sequencing. Cancer Res, 1989, 49: 2 141
, 百拇医药
[2]Tones DD, Scolnick EM, Koller R, et al. ras-related gene sequence identified and isolated from saccharomyces cerevisiae. Nature, 1983, 306(15):707
[3]Serrano M, Hannon G, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature (London), 1993,366: 704
[4]Kamb A, Gruis N, Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994, 264: 436
, 百拇医药
[5]Kendra KL, Katzenllenbogen BS. An evaluation of the involvement of polyamines in modulating MCG-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen . J Steroid Biochem, 1987,28: 123
[6]Lars AL, Paal SA, JФrgen K, et al. A single strand conformation polymorphism/heteroduplex method for detection of mutations in 15 exons of exons KVLQT1 gene , associated with long QT syndrome. Clinica Chimica Acta, 1999, 280:113
[7]Rsseva MG, Janakiev PJ, Kirov SA, et al. A simple method for detectiion of factor V R506Q(Leiden) mutation in dried blood spots. Clinica Chimica Acta, 1999, 284:89
[8]Sambrook J, Fitch EF, Manitatis T. A laboratory manual. New York Cold Spring Harbor Laboratory Press,1990. 467
2000-01-05收稿, 百拇医药
单位：吴逸明(河南医科大学劳动卫生学与卫生毒理学教研室 郑州 450052)；张振中(河南医科大学药学系 郑州 450052)；史香林(美国职业安全卫生研究所病理生理学研究室 摩根墩 WV 26505 USA)；王新朝(河南医科大学劳动卫生学与卫生毒理学教研室 郑州 450052)
关键词：基因；突变；SSCP；K-ras；p16
河南医科大学学报000304 摘要 目的：研究大鼠肺癌组织K-ras和p16 基因突变情况。 方法： 运用聚合酶链反应-单链构象多态性 (PCR-SSCP) 技术分析肺癌组织K-ras和p16 基因突变。结果：未发现正常大鼠肺组织中K-ras和p16 基因突变, 在肿瘤组织中 K-ras和p16均有2例突变发生。结论： K-ras和p16 基因突变与肺癌发生存在一定的相关性.
, http://www.100md.com
分类号 R734.2
Mutation analysis of K-ras and p16 genes in rat lung tissues by SSCP
WU Yiming,WANG Xinchao
(Department of Occupational Health and Health Toxicology, Henan Medical University， Zhengzhou 450052，P.R.China)
ZHANG Zhenzhong,(Department of Pharmacy, Henan Medical University ，Zhengzhou 450052 P.R.China)
SHI Xianglin，(Pathology and Physiology Research, NIOSH, Morgantown, WV 26505, USA)
, 百拇医药
Abstract Aim: To study mutation of K-ras and p16 genes in rat lung tumor tissues. Method: Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to detect the mutation of K-ras and p16 genes in rat lung tissues. Result: For K-ras and p16 gene mutations were not found in normal group and were observed in 2 out of 16 lung tumor tissues. Conclusion: K-ras and p16 genes were correlative to the incidence of lung tumor to some extent.
, http://www.100md.com
Key words gene; mutation; SSCP; K-ras; p16
Epidemiology study has shown that lung cancer is the second commonest cancer and its incidence is increasing dramatically in recent years. It is of great importance to study the mechanism and to find the early biomarkers for lung cancer. Mutations in cellular ras gene have been strongly implicated in various stages of mammalian tumorigenesis. In human tumors, the mutations identified have largely been localized to codon 12,13 or 61 of three ras genes[1,2] : H-ras, K-ras and N-ras. 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[3～5]. Traditionally, mutations of genes were detected by hybridization with DNA probe, allele-specific PCR and restriction fragment length polymorphism (RFLP) and single strand conformational polymorphism (SSCP). SSCP analysis in these PCR-based methods is the simplest and most sensitive. Previous studies showed that gene mutations, such as base substitution, point mutation, deletion and insertion, could lead to conformational change of single-stranded DNA, so the mobility shifts could be observed on the electrophorogram of non-denatured polyacrylamide gel[6,7].
, 百拇医药
1 Materials and methods
1.1 Materials acrylamide, N, N, N', N'-tetramethylenediamine(TEMED) and ammonium peroxydisulfate(APS) were purchased from Sigma Chemical Co. 2-amino-2-(hydroxymethyl)-1,3-propanediol(Tris), boric acid, PCR Markers, Taq DNA polymerase, dimethyl sulfoxide(DMSO), bluephenol, xylene cyanole FF and formamide deionized were obtained from Sino-American Biotechnology Company(SABC). dNTP was from Promega. Primers were synthesized by CyberSyn Biotechnology Corporation (American).
, http://www.100md.com
1.2 Amplification of K-ras and p16 genes by PCR DNA was isolated from normal and tumor lung tissues of rat, by proteinase K digestion and phenol-chloroform extraction using the method of Sambrook[8]. Primers of K-ras gene used were as the following: upstream primer, 5'-ACA/TGT/TCT/AAT/ATA/GTC/AC-3'; downstream prime, 5'-AAA/GAA/TGG/TCC/TGC/ACC/AG-3'. The PCR was performed in a 50 μl buffer containing 50 pmol of each primer, 100 pmol of dATP, dCTP, dGTP, dTTP, 5 μl of 10×PCR buffer, 0.5 μg template. Prior to PCR, the mixture was predenatured at 95 ℃ for 5 min and then 2.0 u polymerase was added to the above solution. All PCR reactions were performed in a DNA cycle of SABC and the reaction mixture was covered by 30 μl wax. The reaction mixture was subject to 30 cycles at 94 ℃ for 60 s, 60 ℃ for 56 s and 72 ℃ for 33 s, the extension time for the last cycle was 5 min at 72 ℃, the length of DNA fragment generated by PCR was 212 bp. Primers of p16 gene used 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'. The PCR reaction constituents were the same as K-ras gene PCR mixture. The reaction mixture was subject to 35 cycles at 94 ℃ for 60 s, 64 ℃ for 50 s and 72 ℃ for 53 s, the extension time for the last cycle was 5 min at 72 ℃, the length of DNA fragment generated by PCR was 240 bp.
, http://www.100md.com
1.3 SSCP by non-denatured vertical slab PAGE 10 μl of PCR products ( K-ras or p16 gene) was added to equal volume of sample buffer containing 20 mmol/L EDTA, 96% formamide deionized, 0.05% bromphenol blue and 0.05% xylene cyanole FF.The above solution was heated at 97 ℃ for 5 min and then immediately chilled in ice water bath for 5 min. 10 μl of the above solution was applied to each lane of non-denatured polyacrylamide gel which crosslinked with N, N'-methylenebisacrylamide(bis) was used at composition of 6%T, 2%C and 5% glycerol for p16 gene. and at composition of 5%T, 1.2% C and 5% glycerol for K-ras gene. Electrophoresis was performed at 200 V for 4 h at 4 ℃ and the gels were stained with silver.
, http://www.100md.com
2 Results and discussion
The amplification of K-ras and p16 genes by PCR generated a DNA fragment of 212 bp and 240 bp, respectively. The PCR products length was identified by comparing with PCR Markers on a conventional 2% agarose gel (data not shown). The amplification of K-ras gene by PCR generated a DNA fragment of 212 bp in the extron 1. Activation of K-ras oncogene is related to the pathogenesis of various human tumors. The incidence of ras gene mutation in the lung tumor is about 20%. Point mutations which led to ras activation were confined to the codon 12, 13 and 61 of the gene. Point mutations in codon 12 are commonly seen. In the present study, the PAGE-SSCP results showed that: no mobility shift appeared in the electrophorogram for the 20 normal tissues, but mobility shift appeared for the samples 10 and 12 in the experiment group (30 cases) in which tumor took place in 16 cases. The migration patterns of K-ras gene from normal lung tissues and lung tumor tissues were shown in Fig 1. There were abnormal band in the lane 1 ( sample 10) and lane 3 ( sample 12 ) in the electrophorogram. This result demonstrated that K-ras gene of samples 10 and 12 mutated in the extron 1.
, 百拇医药
p16 gene (multiple tumor suppression gene) contains3 extrons, which contains 126 bp in the extron 1, 307 bp in the extron 2 and 11 bp in the extron 3 respectively and frequently mutates in extron 2. The vertical slab PAGE-SSCP results showed that: no mobility shift was observed in 20 normal tissues, but mobility shifts were observed in 2 out of 16 tumor samples. The migration patterns of p16 gene from normal lung tissues and tumor lung tissues were shown in Fig 2. In the tumor samples, an abnormal band of allelic single-stranded DNA molecular appeared (lane 2 (sample 10) and lane 7 ( sample 18)) in the electrophorogram. The result demonstrated that p16 gene mutated in samples 10 and 18.
, http://www.100md.com
Fig1 Electrophorogram of SSCP for K-ras gene
N: Normal, M: Mutation
Fig2 Electrophorogram of SSCP for p16 gene
N: Normal, M: Mutation
基金项目：河南省自然科学基金资助项目 204022300
作者简介：吴逸明：男，55岁，教授，博士生导师，研究方向：肺癌的病因学、预防、早期诊断和综合治疗的研究，References
[1]Glenna CB, Peter SR, Lawrence AL. Analysis of c-ki-ras mutations in human colon carcinoma by cell sorting, polymerase chain reaction, and DNA sequencing. Cancer Res, 1989, 49: 2 141
, 百拇医药
[2]Tones DD, Scolnick EM, Koller R, et al. ras-related gene sequence identified and isolated from saccharomyces cerevisiae. Nature, 1983, 306(15):707
[3]Serrano M, Hannon G, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature (London), 1993,366: 704
[4]Kamb A, Gruis N, Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994, 264: 436
, 百拇医药
[5]Kendra KL, Katzenllenbogen BS. An evaluation of the involvement of polyamines in modulating MCG-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen . J Steroid Biochem, 1987,28: 123
[6]Lars AL, Paal SA, JФrgen K, et al. A single strand conformation polymorphism/heteroduplex method for detection of mutations in 15 exons of exons KVLQT1 gene , associated with long QT syndrome. Clinica Chimica Acta, 1999, 280:113
[7]Rsseva MG, Janakiev PJ, Kirov SA, et al. A simple method for detectiion of factor V R506Q(Leiden) mutation in dried blood spots. Clinica Chimica Acta, 1999, 284:89
[8]Sambrook J, Fitch EF, Manitatis T. A laboratory manual. New York Cold Spring Harbor Laboratory Press,1990. 467
2000-01-05收稿, 百拇医药