Single Nucleotide Polymorphisms pp 123-133 | Cite as
Pyrosequencing for SNP Genotyping
Abstract
Pyrosequencing is a real-time DNA sequencing method. It is based on the transformation of pyrophosphates, released during DNA elongation by DNA polymerase, into measurable light. During DNA elongation, a single pyrophosphate molecule is released following incorporation of a single nucleotide. In the pyrosequencing reaction, released pyrophosphates are then rapidly converted by sulfurylase to adenosine triphosphate, which in turn is utilized by luciferase to produce light. Within standardized conditions, this reaction is accomplished in a few milliseconds and the light produced can be registered with a CCD camera. Therefore, it becomes possible to quantitatively measure the nucleotides incorporated. This approach has been automated in different platforms and can be used for a wide variety of applications, such as single-nucleotide polymorphism (SNP) genotyping, DNA sequencing, loss of heterozygosity analysis, and CpG methylation studies. Here we describe the entire process, focusing our attention on SNP genotyping, and giving examples of some other applications.
Key words
Pyrosequencing genotyping sequencing real timeNotes
Acknowledgements
The authors would like to thank Ana Salinas, Maria del Carmen Rivero, and Juan Velasco for their technical support.
References
- 1.Ronaghi, M., Uhlen, M. and Nyren, P. (1998) A sequencing method based on real-time pyrophosphate. Science 281, 363–365.PubMedCrossRefGoogle Scholar
- 2.Ronaghi, M. (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res. 11, 3–11.PubMedCrossRefGoogle Scholar
- 3.Guo, D. C., Qi, Y., He, R., Gupta, P. and Milewicz, D. M. (2003) High throughput detection of small genomic insertions or deletions by Pyrosequencing. Biotechnol. Lett. 25, 1703–1707.PubMedCrossRefGoogle Scholar
- 4.Lavebratt, C. and Sengul, S. (2006) Single nucleotide polymorphism (SNP) allele frequency estimation in DNA pools using Pyrosequencing. Nat. Protoc. 1, 2573–2582.PubMedCrossRefGoogle Scholar
- 5.Loeuillet, C., Weale, M., Deutsch, S., Rotger, M., Soranzo, N., Wyniger, J., Lettre, G., Dupré, Y., Thuillard, D., Beckmann, J. S., Antonarakis, S. E., Goldstein, D. B. and Telenti, A. (2007) Promoter polymorphisms and allelic imbalance in ABCB1 expression. Pharmacogenet. Genomics 17, 951–959.PubMedCrossRefGoogle Scholar
- 6.Lee, E. S., Issa, J. P., Roberts, D. B., Williams, M. D., Weber, R. S., Kies, M. S. and El-Naggar, A. K. (2008) Quantitative promoter hypermethylation analysis of cancer-related genes in salivary gland carcinomas: comparison with methylation-specific PCR technique and clinical significance. Clin. Cancer Res. 14, 2664–2672.PubMedCrossRefGoogle Scholar
- 7.Marsh, S. (2007) Pyrosequencing applications. Methods Mol. Biol. 373, 15–24.PubMedGoogle Scholar
- 8.Alexander, A. M., Pecoraro, C., Styche, A., Rudert, W. A., Benos, P. V., Ringquist, S. and Trucco, M. (2005) SOP3: a web-based tool for selection of oligonucleotide primers for single nucleotide polymorphism analysis by Pyrosequencing. Biotechniques 38, 87–94.PubMedCrossRefGoogle Scholar
- 9.Royo, J. L., Pascual, M. H., Salinas, A., Tello, F. J., Rivero, M. del C., Herrero, E. F., Real, L. M. and Ruiz, A. (2006) Pyrosequencing protocol requiring a unique biotinylated primer. Clin. Chem. Lab. Med. 44, 435–341.Google Scholar
- 10.Royo, J. L., Hidalgo, M. and Ruiz, A. (2007) Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping Nat. Protoc. 2, 1734–1739.PubMedCrossRefGoogle Scholar
- 11.Korshunova, Y., Maloney, R. K., Lakey, N., Citek, R. W., Bacher, B., Budiman, A., Ordway, J. M., McCombie, W. R., Leon, J., Jeddeloh, J. A. and McPherson, J. D. (2008) Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine- methylation patterns obtained from tissue and serum DNA. Genome Res. 18, 19–29.PubMedCrossRefGoogle Scholar
- 12.Pascual, M. H., Royo, J. L., Martínez-Tello, F. J., Crespo, C., Salinas, A., Herrero, E. F., Lopez-García, M., Real, L.M., Ruiz, A. and Ramirez-Lorca, R. (2006) Exploring allelic imbalance within paraffin-embedded tumor biopsies using pyrosequencing technology. Clin. Chem. Lab. Med. 44, 1076–1081.PubMedCrossRefGoogle Scholar