Designing primers against a region of interest (ROI) is the most critical step in obtaining adequate DNA methylation results using PCR-based methods. Several software platforms such as Methyl Primer Express (Applied Biosystems, Foster City, CA), MethPrimer (15), BiSearch (16), MethMaker (17), and MSPprimer (18) have been developed for this purpose. All of these programs allow users to customize primer length, amplicon length, and Tm (melting temperature) differences, as well as enable searches for CpG islands in the input sequence, and identify possible stable primer-dimer or hairpin structures that should be avoided. The advantages and disadvantages of each program are compared in Table 3. Primers should not bind to regions containing common SNPs (19), which can be identified easily using the UCSC Genome Browser (http://genome.ucsc.edu).Table 3.
Because bisulfite treatment decreases DNA sequence complexity, primers have an increased tendency to bind multiple target sequences in converted DNA (18). Therefore, in silico evaluation of primer specificity is a key step during primer design for bisulfite-converted DNA methods. BiSearch software is unique in terms of its ability to find the number of potential matches, including partial matches, for each individual primer in the bisulfite-converted methylated or unmethylated genome and to perform in silico PCR on the bisulfite-converted human genome using any primer pair (16). In our laboratory, we have observed greater success using primers with less than 3000 matches.
At present, the software available for primer design does not account for PCR bias (20-22). When faced with bias, it is important to use additional tools to review the ROI sequence, highlight the CpG and non-CpG cytosines, and design adequate primers. BioWord is a free Microsoft Word plugin that allows manipulation, editing, and processing of DNA sequences and has proven useful for working with sequences prone to PCR bias (23). Another available option is a shareware version of the licensed software FastPCR, which includes a tool for in silico bisulfite conversion of non-CpG cytosines (24).PCR-based techniques Bisulfite sequencing PCR
Bisulfite sequencing PCR (BSP) was the first technique described for analyzing DNA methylation status using PCR (25). The technique consists of PCR amplifying a bisulfite-converted DNA ROI, followed by Sanger sequencing of the product either directly or after cloning into a suitable vector.
Direct-BSP: By comparing sequencing results with the respective reference genomic DNA sequences, direct sequencing of PCR products provides information on the average methylation status for each CpG dinucleotide. Direct-BSP is the shortest form of BSP, but holds several technical challenges inherent in sequencing, such as poor signal quality and artifacts in cytosine signals that may affect electropherogram analysis; it also has a low sensitivity (26). Because of these difficulties, BSP with cloning is more common.
Cloning-based BSP: In cloning-based BSP, PCR products are cloned into a vector and transformed into competent E. coli cells. After expansion and purification of the plasmids, the PCR product inserts are sequenced. The CpG methylation status for each CpG dinucleotide in the ROI is determined by sequencing each expanded clone (27,28). The resulting averages are referred to as DNA-methylation haplotypes. Cloning-based BSP requires at least six sequencing reactions to obtain a sensitivity higher than direct BSP (29), making this an expensive and labor-intensive option that is especially cumbersome for population-based studies.
Digital (single-molecule) BSP: Another BSP option for producing DNA-methylation haplotypes is digital-BSP (22,30). This method requires serial dilution of a DNA template to optimize conditions for PCR amplification of a single converted DNA molecule per reaction tube (via the Poisson distribution), thus avoiding both PCR bias and cloning. Digital-BSP is considered the gold standard for detecting the methylation status of specific loci (22). However, this method is inefficient because 87% of the reactions cannot be analyzed, and 3% are control reactions; thus, useful information is only obtained from the remaining 10% (22). An alternative approach is to use MS-HRM to select the clones for sequencing (31).
Primer design considerations: Methylation independent PCR (MIP) primers should be designed to allow the amplification of bisulfite-converted DNA regardless of methylation status. Primers should also not bind regions containing CpG dinucleotides (Figure 2A) (25) and should flank a sequence of converted DNA containing as many thymines originating from the conversion of non-CpG cytosines as possible (25). Guidelines for designing BSP primers were initially published by Clark et al. in 1994 (25).