A new approach to primer design for the control of PCR bias in methylation studies

被引：96

作者：

Wojdacz T.K. ^{[1
,2
]}

Hansen L.L. ^{[2
]}

Dobrovic A. ^{[1
,3
]}

机构：

[1] Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, St. Andrews Place

[2] Institute of Human Genetics, University of Aarhus, Bartholin Building, DK-8000, Aarhus C

[3] Department of Pathology, University of Melbourne, Parkville

来源：

BMC Research Notes | / 1卷 / 1期

基金：

英国医学研究理事会;

关键词：

Methylation Status; Bisulfite; Prime Design; Methylation Analysis; High Resolution Melting;

D O I：

10.1186/1756-0500-1-54

中图分类号：

学科分类号：

摘要：

Primer design for PCR-based methylation analysis following bisulfite conversion of DNA is considerably more complex than primer design for regular PCR. The choice of the optimal primer set is critical to the performance and correct interpretation of the results. Most methodologies in methylation analysis utilize primers that theoretically amplify methylated and unmethylated templates at the same time. The proportional amplification of all templates is critical but difficult to achieve due to PCR bias favouring the amplification of the unmethylated template. The focus of this brief communication is to point out the important criteria needed for the successful choice of primers that will enable the control of PCR bias in bisulfite based methylation-screening protocols. © 2008 Wojdacz et al; licensee BioMed Central Ltd.

引用

共 16 条

[1]

Dobrovic A., Methods for Analysis of DNA Methylation, Molecular Diagnostics for the Clinical Laboratorian, pp. 149-160, (2005)

[2]

Dahl C., Guldberg P., DNA methylation analysis techniques, Biogerontology, 4, pp. 233-250, (2003)

[3]

Frommer M., McDonald L.E., Millar D.S., Collis C.M., Watt F., Grigg G.W., Molloy P.L., Paul C.L., A Genomic Sequencing Protocol That Yields a Positive Display of 5-Methylcytosine Residues in Individual DNA Strands, Proc Natl Acad Sci USA, 89, pp. 1827-1831, (1992)

[4]

Clark S.J., Harrison J., Paul C.L., Frommer M., High sensitivity mapping of methylated cytosines, Nucleic Acids Res, 22, pp. 2990-2997, (1994)

[5]

Xiong Z., Laird P.W., COBRA: A sensitive and quantitative DNA methylation assay, Nucleic Acids Research, 25, 12, pp. 2532-2534, (1997)

[6]

Eads C.A., Laird P.W., Combined bisulfite restriction analysis (COBRA), Methods Mol Biol, 200, pp. 71-85, (2002)

[7]

Couvert P., Poirier K., Carrie A., Chalas C., Jouannet P., Beldjord C., Bienvenu T., Chelly J., Kerjean A., DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes, Biotechniques, 34, pp. 356-362, (2003)

[8]

Bianco T., Hussey D., Dobrovic A., Methylation-sensitive, single-strand conformation analysis (MS-SSCA): A rapid method to screen for and analyze methylation, Hum Mutat, 14, pp. 289-293, (1999)

[9]

Burri N., Chaubert P., Complex methylation patterns analyzed by single-strand conformation polymorphism, Biotechniques, 26, pp. 232-234, (1999)

[10]

Wojdacz T.K., Hansen L.L., Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay, Biotechniques, 41, (2006)

← 1 2 →