MethPrimer - Design Primers for
Input sequence: A DNA sequence in any format. The sequence should not be edited to mimic bisulfite modification by converting 'C's to 'T'. The program will do the job for you.
Bisulfite sequencing PCR (BSP): DNA is first denatured to create single-stranded DNA and then modified with sodium bisulfite followed by PCR amplification using primers that are specific for the modified DNA but do not contain any CpG sites in their sequence. The resulting PCR product is sequenced directly or after cloning.
Bisulfite restriction PCR: DNA is modification and PCR amplification is performed as stated above. The resulting PCR product is digested with appropriate restriction enzymes.
Methylation-Specific PCR (MSP): DNA is first denatured to create single-stranded DNA and then modified with sodium bisulfite followed by PCR amplification using two pairs of primers, with one pair specific for methylated DNA; the other unmethylated DNA.
CpG island prediction
CpG island prediction: If this option is checked, primers will be picked around the predicted CpG islands. If more than one island is found, any islands will be a potential target region.
Window: The program will slide across input sequence calculating parameters in a window size specified here.
Step: The moving step for the window.
Obs/Exp: Observed/Expected CpG ratio.
GC percentage: Percentage of G plus C.
General Parameters for Primer Selection
Sequence name (optional): Give a name to your sequence.
Target: Specify a region to be flanked by primers. It should not bigger than the maximum product size. You can also mark your source sequence with "[ ]", e.g., ...ATCT[...CCGT...]ATCT...
Excluded regions: The regions should be avoided for primer selection. You can also mark your source sequence with "<" and ">", e.g. ...ATCT<...CCCC...>TCAT..
Number of output pairs: The number of primer pairs to be returned by the program. The default is 5.
Product size: For bisulfite PCR, the product size is usually smaller than that for regular PCR.
Primer size: For bisulfite PCR, the primer size is usually bigger than that for regular PCR.
Primer Poly X: The maximum allowable number of consecutive mononucleotides in a primer sequence except 'T'.
Primer Poly T: The maximum allowable number of consecutive 'T's (or 'A's in a reverse primer) in a primer sequence. The 'T's here includes both original 'T's and non-CpG 'C's which have been converted to 'T'.
Primer non-CpG 'C's: The minimum number of non-CpG 'C's in a primer. This is important for discriminating between the bisulfite-modified DNA and unmodified or incompletely modified DNA.
Product CpGs: The minimum number of CpG sites in the product. This is meaningful for sequencing PCR, since we wish to examine as many CpG sites as possible by one PCR amplification. This constraint is only enforced for BSP primers.
Parameters for MSP primers
3’ CpG constraint: The position of the CpG at a primer’s 3’ end, e.g., a value of "3" means that a CpG site must appear at any of the last 3 bases of the 3' end.
CpG in primer: The minimum number of CpGs in a MSP primer. At least one CpG site is required. If only one CpG occurs in a primer, it must be at the very 3' end.
Max Tm difference: The maximum Tm difference between primer pairs (the methylated and unmethylated). This is useful only if you want to perform methylation-specific PCR and unmethylation-specific PCR using the same cycling conditions.