MethPrimer Help
Input Requirements
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.
PCR Techniques
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
Give a name to your sequence (optional).
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 Tm
Primer melting temperature. The value is usually lower than that for standard PCR.
Primer Poly X
The maximum allowable number of consecutive mononucleotides in a primer sequence except '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.
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.
Parameters for BSP Primers
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.