Warnecke P M, Stirzaker C, Melki J R, Millar D S, Paul C L, Clark S J
Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia, School of Biological Sciences, A12 University of Sydney, NSW 2006, Australia and CSIRO Division of Molecular Science, Sydney.
Nucleic Acids Res. 1997 Nov 1;25(21):4422-6. doi: 10.1093/nar/25.21.4422.
Methylation analysis of individual cytosines in genomic DNA can be determined quantitatively by bisulphite treatment and PCR amplification of the target DNA sequence, followed by restriction enzyme digestion or sequencing. Methylated and unmethylated molecules, however, have different sequences after bisulphite conversion. For some sequences this can result in bias during the PCR amplification leading to an inaccurate estimate of methylation. PCR bias is sequence dependent and often strand-specific. This study presents a simple method for detection and measurement of PCR bias for any set of primers, and investigates parameters for overcoming PCR bias.
基因组DNA中单个胞嘧啶的甲基化分析可通过亚硫酸氢盐处理和目标DNA序列的PCR扩增进行定量测定,随后进行限制性内切酶消化或测序。然而,甲基化和未甲基化的分子在亚硫酸氢盐转化后具有不同的序列。对于某些序列,这可能导致PCR扩增过程中的偏差,从而导致甲基化估计不准确。PCR偏差取决于序列,且通常具有链特异性。本研究提出了一种检测和测量任何引物组PCR偏差的简单方法,并研究了克服PCR偏差的参数。