Summary
Numerous innovative and high-throughput techniques have been established to identify human disease genes. However, DNA sequencing of candidate genes still remains as a major limitation in the identification of causative mutations. Much of this limitation is due to the time and labor needed for the polymerase chain reaction (PCR) optimization and reaction setup. Toward this end, we have established a simplified protocol that utilizes a single PCR amplification condition. PCR purification is accomplished via enzymatic digestion and all products can be sequenced using universal primers. This combination of a single amplification condition, single-step purification, and sequencing setup using universal primers all contribute to a simple and high-throughput mutation screen.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sawcer, S., Ban, M., Maranian, M., Yeo, T.W., Compston, A., Kirby, A., Daly, M.J., De Jager, P.L., Walsh, E., Lander, E.S., (2005) A high-density screen for linkage in multiple sclerosis. Am J Hum Genet 77:454–467
Janecke, A.R., Thompson, D.A., Utermann, G., Becker, C., Hubner, C.A., Schmid, E., McHenry, C.L., Nair, A.R., Ruschendorf, F., Heckenlively, J., Wissinger, B., Nurnberg, P., Gal, A. (2004) Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy. Nat Genet 36:850–854
Puffenberger, E.G., Hu-Lince, D., Parod, J.M., Craig, D.W., Dobrin, S.E., Conway, A.R., Donarum, E.A., Strauss, K.A., Dunckley, T., Cardenas, J.F., Melmed, K.R., Wright, C.A., Liang, W., Stafford, P., Flynn, C.R., Morton, D.H., Stephan, D.A. (2004) Mapping of sudden infant death with dysgenesis of the testes syndrome (SIDDT) by a SNP genome scan and identification of TSPYL loss of function. Proc Natl Acad Sci U S A 101:11689–11694
Sellick, G.S., Garrett, C., Houlston, R.S. (2003) A novel gene for neonatal diabetes maps to chromosome 10p12.1-p13. Diabetes 52:2636–2638
Cvetkovic, D., Pisarcik, D., Lee, C., Hamilton, T.C., Abdollahi, A. (2004) Altered expression and loss of heterozygosity of the LOT1 gene in ovarian cancer. Gynecol Oncol 95:449–455
Koppert, L.B., van der Velden, A.W., van de Wetering, M., Abbou, M., van den Ouweland, A.M., Tilanus, H.W., Wijnhoven, B.P., Dinjens, W.N. (2004) Frequent loss of the AXIN1 locus but absence of AXIN1 gene mutations in adenocarcinomas of the gastro-oesophageal junction with nuclear beta-catenin expression. Br J Cancer 90:892–899
Maris, T., Androulidakis, E.J., Tzagournissakis, M., Papavassiliou, S., Moser, H., Plaitakis, A. (1995) X-linked adrenoleukodystrophy presenting as neurologically pure familial spastic paraparesis [see comments]. Neurology 45:1101–1104
Stephens, L.A., Powell, N.G., Grubb, J., Jeremiah, S.J., Bethel, J.A., Demidchik, E.P., Bogdanova, T.I., Tronko, M.D., Thomas, G.A. (2005) Investigation of loss of heterozygosity and SNP frequencies in the RET gene in papillary thyroid carcinoma. Thyroid 15:100–104
Steinthorsdottir, V., Thorleifsson, G., Reynisdottir, I., Benediktsson, R., Jonsdottir, T., Walters, G.B., Styrkarsdottir, U., Gretarsdottir, S., Emilsson, V., Ghosh, S., (2007) A variant in CDKAL1 influences insulin response and risk of type 2 diabetes. Nat Genet 39:770–775
Klein, R.J., Zeiss, C., Chew, E.Y., Tsai, J.Y., Sackler, R.S., Haynes, C., Henning, A.K., SanGiovanni, J.P., Mane, S.M., Mayne, S.T., Bracken, M.B., Ferris, F.L., Ott, J., Barnstable, C., Hoh, J. (2005) Complement factor H polymorphism in age-related macular degeneration. Science 308:385–389
Sladek, R., Rocheleau, G., Rung, J., Dina, C., Shen, L., Serre, D., Boutin, P., Vincent, D., Belisle, A., Hadjadj, S., Balkau, B., Heude, B., Charpentier, G., Hudson, T.J., Montpetit, A., Pshezhetsky, A.V., Prentki, M., Posner, B.I., Balding, D.J., Meyre, D., Polychronakos, C., Froguel, P. (2007) A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature 445:881–885
Saxena, R., Voight, B.F., Lyssenko, V., Burtt, N.P., de Bakker, P.I., Chen, H., Roix, J.J., Kathiresan, S., Hirschhorn, J.N., Daly, M.J., (2007) Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science 316:1331–1336
Jordan, B., Charest, A., Dowd, J.F., Blumenstiel, J.P., Yeh, R.F., Osman, A., Housman, D.E., Landers, J.E. (2002) Genome complexity reduction for SNP genotyping analysis. Proc Natl Acad Sci U S A 99:2942–2947
Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K., Mattick, J.S. (1991) ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19:4008
Tsang, S., Sun, Z., Stewart, C., Lum, N., Frankenberger, C., Subleski, M., Rasmussen, L., Munroe, D.J. (2004) Development of multiplex DNA electronic microarrays using a universal adaptor system for detection of single nucleotide polymorphisms. Biotechniques 36:682–688
Werle, E., Schneider, C., Renner, M., Volker, M., Fiehn, W. (1994) Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Res 22:4354–4355
Hanke, M., Wink, M. (1994) Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. Biotechniques 17:858–860
Rozen, S., Skaletsky, H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
Kent, W.J., Sugnet, C.W., Furey, T.S., Roskin, K.M., Pringle, T.H., Zahler, A.M., Haussler, D. (2002) The human genome browser at UCSC. Genome Res 12:996–1006
Nickerson, D.A., Tobe, V.O., Taylor, S.L. (1997) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res 25:2745–2751
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Shi, L., Landers, J.E. (2009). High-Throughput Mutation Screening Using a Single Amplification Condition. In: Tainsky, M. (eds) Tumor Biomarker Discovery. Methods in Molecular Biology, vol 520. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-811-9_14
Download citation
DOI: https://doi.org/10.1007/978-1-60327-811-9_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-810-2
Online ISBN: 978-1-60327-811-9
eBook Packages: Springer Protocols