Frequently Asked Questions

MethPrimer is a specialized tool designed for creating primers for methylation analysis using bisulfite conversion. It assists researchers in studying DNA methylation, an important epigenetic modification that affects gene expression.

MethPrimer offers the following capabilities:

• CpG Island Prediction: Identifies regions of the genome with a high frequency of CpG dinucleotides, typically found near gene promoters.
• Bisulfite Sequencing PCR (BSP) Primer Design: Designs primers that amplify bisulfite-modified DNA while being unbiased towards methylated or unmethylated DNA.
• Methylation-Specific PCR (MSP) Primer Design: Creates two pairs of primers for MSP, one pair specific for methylated DNA, and the other for unmethylated DNA.

Bisulfite-conversion-based methylation PCR primer design is more complex than standard PCR for several reasons:

• Sequence Modification: Bisulfite treatment converts non-CpG cytosines to thymines, leading to low GC content and long stretches of 'T's.
• Specific Constraints: Bisulfite PCR primers have unique requirements, such as excluding CpG sites in BSP and having at least one CpG in MSP primers.

These factors mean that finding primer pairs is not always guaranteed.

When no primers are found, you should try:

• Relaxing Parameters: Loosen restrictions on product CpG count (BSP), primer CpG count (MSP), non-CpG 'C's, Tm difference (MSP), and 3' CpG constraint (MSP). Experiment systematically.
• Specifying a Target Region: Define your region of interest within the input sequence. This can sometimes improve primer selection results.
• Reviewing Input Sequence: Ensure the quality of your input sequence, and make sure it is the correct one for your analysis.

MethPrimer employs a multi-step process for selecting appropriate primers:

• Bisulfite Conversion Simulation: It converts non-CpG 'C's to 'T's to create the modified sequence, which is used for all primer design. For MSP, both a modified sequence and an "unmethylated sequence" (which treats all cytosines as methylated) are used.
• Oligo Testing: Tests all possible short sequences (oligos) within the sequence against multiple parameters (GC content, Tm, non-CpG 'C's, CpGs in primers)
• Pair Matching: Combines two oligos (upstream and downstream) and tests them against product parameters (product size, Tm, location relative to the target and CpG islands). For MSP it will look for two pairs of primers that match the methylated and unmethylated forms.
• Parameter Check: Check the combined primer pairs against user-provided constraints.
• Results: Returns primer pairs that best meet all defined constraints and requirements.

CpG refers to a cytosine ('C') nucleotide followed by a guanine ('G') nucleotide in the DNA sequence. The 'p' represents the phosphodiester bond linking the two bases. CpG dinucleotides are critical in methylation studies, which may affect gene expression.

MethPrimer applies many rules during primer design. See our Help Page for full details on each parameter and rule used for primer selection.

Yes, MethPrimer is provided free of charge to all users. We strive to make essential scientific tools available without financial barriers.

We value your feedback to help us improve MethPrimer. If you encounter any issues or have suggestions for new features, please reach out to us using the contact form available on our website. Your input is invaluable in enhancing our services.

When using MethPrimer for your work, please cite the original publication:

MethPrimer builds upon the foundational work of Primer3, a well-known primer design program developed by Steve Rozen and Helen J. Skaletsky at the Whitehead Institute for Biomedical Research.