Abstract
A novel PCR method using confronting two‐pair primers, named PCR‐CTPP, is introduced to detect a single nucleotide polymorphism (base X or Y). One primer for the X allele is set to include X'at the 3′end (antisense), where X'is the antisense of X, with the counterpart sense primer upstream. For the Y allele, a sense primer including Y at the 3′end is set, with the antisense primer downstream. One common band and one specific band for each allele are amplified, which allows genotyping directly by electrophoresis. This method is exemplified by application to the polymorphisms of beta‐adrenoceptor 2 and interleukin 1B. It is simpler than PCR‐RFLP (restriction fragment length polymorphism), which requires incubation with a restriction enzyme, and is suitable for genotyping in studies of genetic epidemiology involving hundreds of samples.
Keywords: PCR, Confronting two‐pair primers, Polymorphism
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REFERENCES
- 1. ) Kawajiri , K. and Fujii‐Kuriyama , Y.P450 and human cancer . Jpn. J. Cancer Res. , 82 , 1325 – 1335 ( 1991. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. ) Hussain , S. P. and Harris , C. C.Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes . Cancer Res. , 58 , 4023 – 4037 ( 1998. ). [PubMed] [Google Scholar]
- 3. ) Perera , F. P.Molecular epidemiology: on the path to prevention ? J. Natl. Cancer Inst. , 92 , 602 – 612 ( 2000. ). [DOI] [PubMed] [Google Scholar]
- 4. ) Khoury , M. J. and Flanders , W. D.Nontraditional epidemiologic approaches in the analysis of gene‐environment interaction: case‐control studies with no controls! Am. J. Epidemiol. , 144 , 207 – 213 ( 1996. . [DOI] [PubMed] [Google Scholar]
- 5. ) Hamajima , N. , Yuasa , H. , Matsuo , K. and Kurobe , Y.Detection of gene‐environment interaction by case‐only studies . Jpn. J. Clin. Oncol. , 29 , 490 – 493 ( 1999. ). [DOI] [PubMed] [Google Scholar]
- 6. ) Erlich , H. A. , Gelfand , D. and Sninsky , J. J.Recent advances in the polymerase chain reaction . Science , 252 , 1643 – 1651 ( 1991. ). [DOI] [PubMed] [Google Scholar]
- 7. ) Orita , M. , Iwahana , H. , Kanazawa , H. , Hayashi , K. and Sekiya , T.Detection of polymorphisms of human DNA by gel electrophoresis as single‐strand conformation polymorphisms . Proc. Natl. Acad. Sci. USA , 86 , 2766 – 2770 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. ) Brown , P. O. and Botstein , D.Exploring the new world of the genome with DNA microarrays . Nat. Genet. , 21 , 33 – 37 ( 1999. ). [DOI] [PubMed] [Google Scholar]
- 9. ) Matsuo , K. , Hamajima , N. , Tominaga , S. , Suzuki , T. , Nakamura , T. , Matsuura , A. and Kitayama , K.Helicobacter pylori IgG antibody test established in the United States showed a substantially lower sensitivity for Japanese population . Am. J. Gastroenteol. ( in press ). [DOI] [PubMed] [Google Scholar]
- 10. ) Hamajima , N. , Iwata , H. , Obata , Y. , Matsuo , K. , Mizutani , M. , Iwase , T. , Miura , S. , Okuma , K. , Ohashi , K. and Tajima , K.No association of the 5′promoter region polymorphism of CYP17 with breast cancer risk in Japan . Jpn. J. Cancer Res. , 91 , 880 – 885 ( 2000. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. ) Large , V. , Hellstrom , L. , Reynisdottir , S. , Lonnqvist , F. , Eriksson , P. , Lannfeld , L. and Arner , P.Human beta‐2 adrenoceptor gene polymorphisms are highly frequent in obesity and associate with altered adipocyte beta‐2 adrenoceptor function . J. Clin. Invest. , 100 , 3005 – 3013 ( 1997. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. ) El‐Omar , E. M. , Carrington , M. , Chow , W.‐H. , McColl , K. , Bream , J. H. , Young , H. A. , Herrera , J. , Lissowska , J. , Yuan , C.‐C. , Rothman , N. , Lanyon , G. , Martin , M. , Fraumeni , J. F. , Jr. and Rabkin , C. S.Interleukin‐1 polymorphisms associated with increased risk of gastric cancer . Nature , 404 , 398 – 402 ( 2000. ). [DOI] [PubMed] [Google Scholar]
- 13. ) Blum , M. , Demierre , A. , Grant , D. M. , Heim , M. and Meyer , U. A.Molocular mechanism of slow acetylation of drugs and carcinogens in humans . Proc. Natl. Acad. Sci. USA , 88 , 5237 – 5241 ( 1991. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. ) Takeda , S. , Ichii , S. and Nakamura , Y.Detection of K‐ras mutation in sputum by mutant‐allele specific amplification (MASA) . Hum. Mutat. , 2 , 112 – 117 ( 1993. ). [DOI] [PubMed] [Google Scholar]
- 15. ) Bailey , L. R. , Roodi , N. , Verrier , C. S. , Yee , C. J. , Dupont , W. D. and Parl , F. F.Breast cancer and CYP1A1, GSTM1, and GSTT1 polymorphisms: evidence of a lack of association in Caucasians and African Americans . Cancer Res. , 58 , 65 – 70 ( 1998. ). [PubMed] [Google Scholar]