MethPrimer - Design Primers for
Methylation PCRs
F A Q
MethPrimer is a program for designing bisulfite-conversion-based Methylation PCR Primers.
Here are things that MethPrimer can do: CpG island prediction. CpG islands are short stretches of DNA in which the frequency of the CpG sequences is higher than in other regions. The accepted definition of a CpG island is a region of DNA greater than 200 bp, with a GC content above 0.5 and an observed/expected CpG (Obs/Exp) ratio above 0.6. CpG islands are often located surrounding the promoter of housekeeping genes or other genes frequently expressed in cells. In normal situation, CpGs in CpG islands are not methylated except those associated with imprinted genes and on inactivated X chromosome in female. By contrast, in some diseases such as cancer, CpG islands can become methylated, leading to silencing of their downstream genes. Mapping of the methylation patterns in CpG islands has become an important tool for understanding both normal and aberrant gene expression. Hence it is helpful to incorporate the CpG island information into primer design for various methylation mapping PCRs. MethPrimer can search for CpG islands in an input sequence and subsequently design primers around the predicted islands.
Design primer for bisulfite sequence PCR (BSP). For BSP, DNA is first denatured to created single stranded DNA and then modified with sodium bisulfite, followed by PCR amplification using primers specific for the modified DNA, but with no bias for methylated or unmethylated DNA (primers contain no CpG 'C's). The resulting PCR products can then be used for sequencing or restriction digestion to map the methylation status of the CpG sites within the amplified region.
Design primer for methylation-specific PCR (MSP). For MSP, DNA is modified as stated above followed by PCR amplification in two separate reactions using two pairs of primers, with one specific for the modified and methylated DNA (M pair), the other specific for the modified and unmethylated DNA (U pair). The resulting PCR product is analyzed by agarose electrophoresis. Amplification from the M pair and the U pair indicates methylation and unmethylation respectively. If both pairs show amplification from a DNA sample, the sample is considered to be partially methylated.
Designing primers for bisulfite-conversion-based methylation PCR is very different from that for standard PCR, because, first, the input sequence is modified to convert non-CpG ‘C’s to ‘T’, resulting in a low GC content with long stretches of ‘T’s in the input sequence, which is not ideal for primer selection; second, many extra constraints are applied to primer selection in addition to those for standard PCR. For example, bisulfite sequencing PCR primers should contain no CpG sites in their sequences, but should span a region with the maximum number of CpG sites; while for MSP, a primer should contain at least one CpG site in its sequence. Therefore, it is not uncommon to get no primer hit for an input sequence.
There are several measures you can take to force the program to pick primers without significantly sacrificing primer quality.
Try to relax some parameters such as the number of CpGs in the product (for BSP) and in a primer (for MSP), the number of non-CpG ‘C’s in a primer (for both BSP and MSP), the Tm difference between two sets of primers (for MSP), the 3’ end CpG constraint (for MSP).
Try to specify a target region of interest if you have not done so. Sometimes, this yields better results.
MethPrimer first takes your input sequence which can be in any format (No sequence editing is needed before input) and internally converts non-CpG ‘C’s in the sequence to ‘T’. The resulting sequence is called the "modified sequence". For MSP primer, in addition to the version of the "modified sequence", another version called the “unmethylated sequence” is also stored internally. The program will then test every substring (oligo) of the input sequence against various parameters, such as GC content, complementarity, Tm, number of non-CpG ‘C’s, number of CpGs (for MSP), and position of 3’ end CpGs. Then any combinations of two oligos (upstream and downstream) will be tested for pair parameters such as product size, product Tm, pair complementarities, position in relation to target region, excluded region and CpG islands, and number of CpG sites in the product. For MSP, additional procedures are performed to match two pairs of primers to make a set. The methylation-specific and unmethylation-specific pair in a set should contain the same CpG sites in their sequence, and preferably have similar Tm values. Finally, the program returns the best primers that meet all constraints.
CpG refers to a ‘C’ nucleotide immediately followed by a ‘G’. The 'p' in 'CpG' simply refers to the phosphate group linking the two bases.
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