SpringerProtocols: Abstract: 12. Genotyping SNPs Using a UV-Photocleavable Oligonucleotide in MALDI-TOF MS

12. Genotyping SNPs Using a UV-Photocleavable Oligonucleotide in MALDI-TOF MS

By: Peter M. Vallone², Kristina Fahr³, Markus Kostrzewa³

Abstract

Full Text

Download PDF (142K)

Matrix-assisted laser desorption time-of-flight mass spectrometry coupled with allelespecific primer extension is a proven method for typing single nucleotide polymorphisms (SNPs). A novel modification upon this methodology is the incorporation of a photocleavable linker within the extension primer. After completion of the primer extension reaction, photocleavage of the extension products results in two deoxyribonucleic acid (DNA) fragments of lower mass. Typically, the smaller cleavage product, which contains the genotyping information, is in the range of 1000-3000 Daltons. The decrease in primer mass allows for higher sensitivity in mass spectrometric measurement and increases the potential for higher levels of multiplexing.The disturbing mass spectrometric analysis peaks caused by salt adducts and doubly charged ions are diminished when analyzing lower-mass DNA fragments. Here, we illustrate the methodology for using photocleavable modified extension primers for detection of SNPs located on the Y chromosome.

Genomic templates were prepared from anonymous male donors. Five regions of the Y chromosome containing the SNP markers M9, M42, M45, M89, and M96 were amplified by polymerase chain reaction, treated with shrimp alkaline phosphatase, and subjected to primer extension reactions using primers containing a photocleavable building block at specific sites. After elongation, the extension primers were desalted and subjected to ultraviolet irradiation to cleave the products at the photocleavable site. Subsequently, the small fragments derived from the 3′' ends of the molecules containing the genotype information were analyzed by matrix-assisted laser desorption ionization time of-flight using a 3-hydroxypicolinic acid matrix.

Affiliation(s): (2) Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD
(3) Bruker Daltonik GmbH, Leipzig, Germany

Book Title: Forensic DNA Typing Protocols

Series: Methods in Molecular Biology | Volume: 297 | Pub. Date: Nov-30-2004 | Page Range: 169-178 | DOI: 10.1385/1-59259-867-6:169

Subject: Genetics/Genomics

Key Words: MALDI-TOF MS - primer extension - multiplex - SNP - PCR - genotype - Y chromosome

References

Download References

1.	Little, D. P., Braun, A., Darnhofer-Demar, B., Frilling, A., Li, Y., McIver, R. T., Jr., et al. (1997) Detection of RET proto-oncogene codon 634 mutations using mass spectrometry. J. Mol. Med. 75, 745–750.

2.	Haff, L. A., and Smirnov, I. P. (1997) Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry. Genome Res. 7, 378–388.

3.	Griffin, T. J. and Smith, L. M. (2000) Single-nucleotide polymorphism analysis byMALDI-TOF mass spectrometry. Trends Biotechnol. 18, 77–84.

4.	Pusch, W., Wurmbach, J. H., Thiele, H., and Kostrzewa, M. (2002) MALDI-TOF mass spectrometry-based SNP genotyping. Pharmacogenomics 3, 537–548.

5.	Sauer, S., Lechner, D., Berlin, K., Lehrach, H., Escary, J. L., Fox, N., and Gut, I. G. (2000) A novel procedure for efficient genotyping of single nucleotide polymorphisms. Nucleic Acids Res. 28, E13.

6.	Sauer, S., Lechner, D., Berlin, K., Plancon, C., Heuermann, A., Lehrach, H., et al. (2000) Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay. Nucleic Acids Res. 28, E100.

7.	Sauer, S., Lehrach, H., and Reinhardt, R. (2003) MALDI mass spectrometry analysis of single nucleotide polymorphisms by photocleavage and charge-tagging. Nucleic Acids Res. 31, E63.

8.	Sauer, S., and Gut, I. G. (2003) Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 17, 1265–1272.

9.	Jobling, M. A., and Tyler-Smith, C. (2003) The human Y chromosome: an evolutionary marker comes of age. Nat. Rev. Genet. 4, 598–612.

10.	Underhill, P. A., Shen, P., Lin, A. A., Jin, L., Passarino, G., Yang, W. H., et al. (2000) Y chromosome sequence variation and the history of human populations. Nat. Genet. 26, 358–361.

11.	Butler, J. M., Devaney, J. M., Marino, M. A., and Vallone, P. M. (2001) Quality control of PCR primers used in multiplex STR amplification reactions. Forensic Sci. Int. 119, 87–96.

12.	Ordoukhanian, P., and Taylor, J-S. (1995) Design and synthesis of a versatile photocleavable DNA building block. Application to phototriggered hybridization. J. Am. Chem. Soc. 117, 9570–9571.

13.	Wu, K. J., Steding, A., and Becker, C. H. (1993) Matrix-assisted laser desorption time-of-flight mass spectrometry of oligonucleotides using 3-hydroxypicolinic acid as an ultraviolet-sensitive matrix. Rapid Commun. Mass Spectrom. 7, 142–146.

14.	Chiu, N. H., Tang, K., Yip, P., Braun, A., Koster, H., and Cantor, C. R. (00) Mass spectrometry of single-stranded restriction fragments captured by an undigested complementary sequence. Nucleic Acids Res. 28, E31.

15.	Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T.J. (ed.) (1990) PCR Protocols A Guide to Methods and Application, Academic Press, San Diego, CA.

16.	Kaderali, L., Deshpande, A., Nolan, J. P., and White, P. S. (03) Primer-design for multiplexed genotyping. Nucleic Acids Res. 31, 1796–1802.

17.	Shaler, T. A., Wickham, J. N., Sannes, K. A., Wu, K. J., and Becker, C. H. (1996) Effect of impurities on the matrix-assisted laser desorption mass spectra of singlestranded oligodeoxynucleotides. Anal. Chem. 68, 576–579.

Comments (Loading...)

Post comment/View all comments