# MethPrimer

MethPrimer is a free, web-based program for designing primers used in PCR-based analysis of DNA methylation. It is specifically designed to work with bisulfite-converted DNA, a common technique in epigenetics research. MethPrimer supports the design of primers for methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). It also includes a feature for predicting CpG islands. The program was created by Dr. Long-Cheng Li and is currently maintained by Ractigen Therapeutics, a biotechnology company founded by Dr. Li in 2017.

## History

MethPrimer was developed by Dr. Long-Cheng Li in 2002 while he was a research scientist at the University of California, San Francisco (UCSF). The program was created to address the growing need for a specialized tool to design primers for bisulfite-based methylation studies, a technique that was becoming increasingly important in epigenetics research. The original MethPrimer program was described in a Bioinformatics journal article published in 2002. The program was originally hosted on the UCSF Li Lab website and was later moved to its own domain, methprimer.com, and is maintained by Ractigen Therapeutics.

## Features

MethPrimer offers several key features for methylation analysis:
* Methylation-Specific PCR (MSP) Primer Design: MethPrimer designs two pairs of primers for MSP: one pair specific to methylated DNA and another pair specific to unmethylated DNA. This allows researchers to determine the methylation status of a specific DNA region.
* Bisulfite Sequencing PCR (BSP) Primer Design: MethPrimer designs primers that amplify bisulfite-converted DNA without bias towards methylated or unmethylated sequences. These primers are used for sequencing-based methylation analysis, providing a detailed view of methylation patterns.
* CpG Island Prediction: MethPrimer can predict CpG islands within a given DNA sequence. CpG islands are regions with a high frequency of CpG dinucleotides, often associated with gene promoters and regulatory elements. The prediction is based on parameters such as GC content, observed/expected CpG ratio, and window size.
* Input Flexibility: Users can input DNA sequences in various standard formats. The program handles the bisulfite conversion simulation internally, so users do not need to pre-edit their sequences.
* Output Formats: MethPrimer provides results in both text and graphical formats, making it easy to visualize primer locations and CpG island predictions.

## Algorithm

MethPrimer's primer design process involves several steps:
1. Bisulfite Conversion Simulation: The input DNA sequence is computationally "converted" to simulate the effects of bisulfite treatment. Non-CpG cytosines are changed to thymines. For MSP, both a fully converted sequence (representing unmethylated DNA) and a sequence where all cytosines are protected (representing methylated DNA) are generated. 2. Oligo Testing: All possible short oligonucleotide sequences (potential primers) within the converted sequence(s) are evaluated based on various parameters, including:
* GC content
* Melting temperature (Tm)
* Number of non-CpG cytosines
* Presence and location of CpGs (for MSP primers)
* Maximum number of consecutive mononucleotides (Poly X and Poly T rules)
3. Primer Pair Matching: Potential upstream and downstream primers are paired and evaluated based on:
* Product size
* Melting temperature difference between primers (especially for MSP)
* Location relative to the target region and predicted CpG islands
4. Parameter Filtering: Primer pairs are filtered based on user-defined constraints, such as product size range, primer size range, Tm range, and specific requirements for BSP and MSP primers.
5. Output: The program returns a list of primer pairs that best meet the specified criteria.

## Impact and usage

MethPrimer has been widely used in the scientific community and has been cited in over 2,900 peer-reviewed publications as of 2025. It is considered a standard tool in the field of epigenetics research, particularly for studies involving DNA methylation. The program's longevity and continued use demonstrate its utility and reliability.