Browse > Article
http://dx.doi.org/10.5483/BMBRep.2013.46.5.155

Detection of rare point mutation via allele-specific amplification in emulsion PCR  

Cheng, Changming (National Engineering Center for Biochip at Shanghai)
Zhou, Yin (National Engineering Center for Biochip at Shanghai)
Yang, Chao (National Engineering Center for Biochip at Shanghai)
Chen, Juan (National Engineering Center for Biochip at Shanghai)
Wang, Jie (National Engineering Center for Biochip at Shanghai)
Zhang, Jie (National Engineering Center for Biochip at Shanghai)
Zhao, Guoping (National Engineering Center for Biochip at Shanghai)
Publication Information
BMB Reports / v.46, no.5, 2013 , pp. 270-275 More about this Journal
Abstract
It is essential to analyze rare mutations in many fields of biomedical research. However, the detection of rare mutations is usually failed due to the interference of predominant wild-type DNA surrounded. Herein we describe a sensitive and facile method of detecting rare point mutation on the basis of allele-specific amplification in emulsion PCR. The identification and selective amplification of rare mutation are accomplished in one-pot reaction. The allele-specific primers coupled on magnetic beads allow the exclusive amplification and enrichment of the mutant amplicons. The productive beads bearing mutant amplicons are subsequently stained with the fluorescent dyes. Thus, the rare point mutations with a percentage as low as 0.1%, can be detected by fluorescent analysis. The relative percentages of mutation among different samples can be roughly accessed by counting the fraction of fluorescent positive beads through flow cytometry.
Keywords
Allele-specific amplification; Emulsion PCR; Rare mutation; TP53 gene;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Pao, W. and Ladanyi, M. (2007) Epidermal growth factor receptor mutation testing in lung cancer: searching for the ideal method. Clin. Cancer Res. 13, 4954-4955.   DOI   ScienceOn
2 Williams, R., Peisajovich, S., Miller, O., Magdassi, S., Tawfik, D. and Griffiths, A. (2006) Amplification of complex gene libraries by emulsion PCR. Nat. Methods 3, 545-550.   DOI   ScienceOn
3 Liu, Q. and Sommer, S. (2004) Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues. Biotechniques 36, 156-167.
4 Endo, K., Konishi, A., Sasaki, H., Takada, M., Tanaka, H., Okumura, M., Kawahara, M., Sugiura, H., Kuwabara, Y. and Fukai, I. (2005) Epidermal growth factor receptor gene mutation in non-small cell lung cancer using highly sensitive and fast TaqMan PCR assay. Lung Cancer 50, 375-384.   DOI   ScienceOn
5 Zhou, C., Ni, J. and Zhao, Y. (2006) Rapid detection of epidermal growth factor receptor mutations in non-small cell lung cancer using real-time polymerase chain reaction with TaqMan-MGB probes. Cancer J. 12, 33-39.   DOI
6 Dunbar, S. A. (2006) Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection. Clin. Chim. Acta. 363, 71-82.   DOI   ScienceOn
7 Frayling, I. (2002) Methods of molecular analysis: mutation detection in solid tumours. Mol. Pathol. 55, 73.   DOI
8 Dutt, A. and Beroukhim, R. (2007) Single nucleotide polymorphism array analysis of cancer. Curr. Opin. Oncol. 19, 43-49.   DOI   ScienceOn
9 Thelwell, N., Millington, S., Solinas, A., Booth, J. and Brown, T. (2000) Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. 28, 3752.   DOI
10 Hayden, E. C. (2009) Personalized cancer therapy gets closer. Nature 458, 131.   DOI   ScienceOn
11 Fleischhacker, M. and Schmidt, B. (2010) Free circulating nucleic acids in plasma and serum (CNAPS) -- Useful for the detection of lung cancer patients? Cancer Biomark 6, 211-219.   DOI
12 Gormally, E., Caboux, E., Vineis, P. and Hainaut, P. (2007) Circulating free DNA in plasma or serum as biomarker of carcinogenesis: Practical aspects and biological significance. Mutat. Res. Rev. Mutat. 635, 105-117.   DOI   ScienceOn
13 Schwarzenbach, H., Hoon, D. S. B. and Pantel, K. (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11, 426-437.   DOI   ScienceOn
14 Sirera, R., Bremnes, R. M., Cabrera, A., Jantus-Lewintre, E., Sanmartín, E., Blasco, A., del Pozo, N., Rosell, R., Guijarro, R. and Galbis, J. (2011) Circulating DNA is a useful prognostic factor in patients with advanced non-small cell lung cancer. J. Thorac. Oncol. 6, 286.   DOI   ScienceOn
15 Mulcahy, H. E., Croke, D. T. and Farthing, M. J. (1996) Cancer and mutant DNA in blood plasma. Lancet 348, 628.   DOI   ScienceOn
16 Diehl, F., Schmidt, K., Choti, M. A., Romans, K., Goodman, S., Li, M., Thornton, K., Agrawal, N., Sokoll, L., Szabo, S. A., Kinzler, K. W., Vogelstein, B. and Diaz, L. A. Jr. (2008) Circulating mutant DNA to assess tumor dynamics. Nat. Med. 14, 985-990.   DOI   ScienceOn
17 Diehl, F., Li, M., Dressman, D., He, Y., Shen, D., Szabo, S., Diaz, L. A. Jr., Goodman, S. N., David, K. A., Juhl, H., Kinzler, K. W. and Vogelstein, B. (2005) Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc. Natl. Acad. Sci. U.S.A. 102, 16368-16373.   DOI   ScienceOn
18 Diehl, F., Li, M., He, Y., Kinzler, K. W., Vogelstein, B. and Dressman, D. (2006) BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat. Methods 3, 551-559.   DOI   ScienceOn
19 Bortolin, S. (2009) Multiplex genotyping for thrombophilia-associated SNPs by universal bead arrays. Methods Mol. Biol. 496, 59-72.
20 Dressman, D., Yan, H., Traverso, G., Kinzler, K. W. and Vogelstein, B. (2003) Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. U.S.A. 100, 8817-8822.   DOI   ScienceOn
21 Little, S. (1995) Amplification-refractory mutation system (ARMS) analysis of point mutations. Curr. Protoc. Hum. Genet. 9.8, 1-12.
22 Newton, C., Graham, A., Heptinstall, L., Powell, S., Summers, C., Kalsheker, N., Smith, J. and Markham, A. (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503.   DOI   ScienceOn