Singal R, Grimes S R
Overton Brooks Veterans Administration Medical Center, Shreveport, LA, USA.
Biotechniques. 2001 Jan;30(1):116-20. doi: 10.2144/01301bc02.
Cytosine methylation at CpG dinucleotides is an important control mechanism in development, differentiation, and neoplasia. Bisulfite genomic sequencing and its modifications have been developed to examine methylation at these CpG dinucleotides. To use these methods, one has to (i) manually convert the sequence to that produced by bisulfite conversion and PCR amplification, taking into account that cytosine residues at CpG dinucleotides may or may not be converted depending on their methylation status, (ii) identify relevant restriction sites that may be used for methylation analysis, and (iii) conduct similar steps with the other DNA strand since the two strands of DNA are no longer complementary after bisulfite conversion. To automate these steps, we have developed a macro that can be used with Microsoft Word. This macro (i) converts genomic sequence to modified sequence that would result after bisulfite treatment facilitating primer design for bisulfite genomic sequencing and methylation-sensitive PCR assay and (ii) identifies restriction sites that are preserved in bisulfite-converted and PCR-amplified product only if cytosine residues at relevant CpG dinucleotides are methylated (and thereby not converted to uracil) in the genomic DNA.
CpG二核苷酸处的胞嘧啶甲基化是发育、分化和肿瘤形成过程中的一种重要调控机制。亚硫酸氢盐基因组测序及其改进方法已被开发出来，用于检测这些CpG二核苷酸处的甲基化情况。要使用这些方法，必须做到以下几点：（i）手动将序列转换为由亚硫酸氢盐转化和PCR扩增产生的序列，要考虑到CpG二核苷酸处的胞嘧啶残基可能会根据其甲基化状态而被转化或未被转化；（ii）识别可用于甲基化分析的相关限制性酶切位点；（iii）对另一条DNA链进行类似步骤，因为亚硫酸氢盐转化后DNA的两条链不再互补。为了实现这些步骤的自动化，我们开发了一个可与Microsoft Word一起使用的宏。这个宏（i）将基因组序列转换为亚硫酸氢盐处理后会产生的修饰序列，便于进行亚硫酸氢盐基因组测序和甲基化敏感PCR分析的引物设计；（ii）识别仅在基因组DNA中相关CpG二核苷酸处的胞嘧啶残基被甲基化（因此未转化为尿嘧啶）时，才会保留在亚硫酸氢盐转化和PCR扩增产物中的限制性酶切位点。
