Word TM Macro for Analysis of Cytosine Methylation by the Bisulfite Deamination Reaction

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® WordTM. 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. INTRODUCTION Cytosine methylation at CpG dinucleotides has increasingly been recognized as an important control mechanism in development, differentiation, and neoplasia. Frommer et al. (1) introduced a procedure based on bisulfateinduced oxidative deamination of genomic DNA under conditions in which cytosine is converted to uracil and 5methyl cytosine (5-mC) remains unchanged. The bisulfite-treated target sequence is then amplified by PCR using strand-specific primers. Upon sequencing of the amplified DNA, all uracil and thymine residues become detectable as thymine, and only 5-mC residues amplify as cytosines. This method is presently the method of choice for the detailed analysis of 5-mC in any given genomic target sequence (6,7,10), and it has been used it in our laboratory to examine the methylation status an avian embryonic globin gene and of the histone H1t promoter (6,9). A number of rapid methods to detect 5mC have been developed based on the bisulfite deamination reaction in combination with PCR amplification. These are suitable for examining limited numbers of CpG dinucleotides and include PCR amplification with primers specific for methylated or unmethylated alleles (3) or identification of restriction sites in PCR-amplified product (4,10). However, to be able to use these methods, one must conduct a time-consuming manual analysis of the sequence of interest. There is a need for a computer program or macro to automate this process. Programs such as MethTools have been written to help design and analyze DNA using the bisulfite genomic sequencing technique (2). However, the programs are written in PERL 5 or C++. To run the programs, you must have a computer with a UNIX® operating system. Investigators that may not have easy access to this operating system most likely do have access to Microsoft® WordTM and other Microsoft Office® applications. Visual Basic® for Applications (VBA) programming language is included with these applications. Therefore, there is a need for a macro that can be used with Microsoft Word to help in DNA analysis for the bisulfite genomic sequencing technique and its modifications. In this paper, we present such a macro. MATERIALS AND METHODS Microsoft VBA supplied with Microsoft Word for Office 2000 was used to prepare this macro. The Macro was tested with Microsoft Word for Office 97 running under Windows 95 and Word for Office 2000 running under Windows 98 on an IBM-compatible computer and with Microsoft Word 98 for the Macintosh on a Power Macintosh G3 running OS 8.1. To run this macro, you need a copy of Microsoft Word running on your computer, and you need to type in or paste in the DNA sequence to be analyzed. You may obtain the source code for the macro as an e-mail attachment from the authors. To incorporate the macro 116 BioTechniques Vol. 30, No. 1 (2001) >>>>>>>>>>>>>>>>>> Microsoft® WordTM Macro for Analysis of Cytosine Methylation by the Bisulfite Deamination Reaction BioTechniques 30:116-120 (January 2001) into Word for Office 2000 on an IBMcompatible computer, you should open Microsoft Word, click the Tools Macro menu item, and click Visual Basic Editor. In the Visual Basic Editor, click File and then click Import File. Click on the macro file name and click the Open button to import the macro. The file is supplied with the extension “.bas” but it may also be supplied as a text file with the extension “.txt”. Both file types can be used. To incorporate the macro into Word on the Macintosh, open the macro text file as a Word file. Click Edit, click Select All, and then click Edit Copy. Click Tools, click Macro, click Macros, and type “Bisulfite” in the Macros Name box. Click Create, click Paste to insert the macro, and click Save Normal. Click File Close and Return to Word. The macro named Bisulfite is then available to analyze your DNA sequence. A macro button can be added to the menu bar to shorten this process to two mouse clicks. To add the button on the PC, click the Tools Customize menu item. Click the Commands tab and click tools in the left window. Scroll down and hold down the left mouse button on the macro button (a filled triangle pointing to the right). Drag the symbol to the menu bar beside Help. Right click on the symbol, click on the “image and text” choice, and close the Customize window. You can click on the new Macros menu item to display the macro list and then click Bisulfite. VBA that is built into Word can be used to modify this macro or to write additional applications. Repetitive tasks such as Edit (Find and Replace) that we use to study bisulfite-modified DNA sequences, as described here, can be automated by writing macros using VBA. Therefore, the tools for the use and modification of this macro are available at little expense. RESULTS AND DISCUSSION