Primer design tasks

BSP: DNA is first treated with sodium bisulfite to convert cytosine to uridine while leaving 5mC unchanged. The converted DNA is then amplified by PCR using primers that are specific for the converted DNA but do not contain any CpG Cs in their sequences. The resulted PCR product is sequenced directly or after cloning.

Methylight BSP: DNA is first treated with sodium bisulfite to convert cytosine to uridine while leaving 5mC unchanged. The converted DNA is then amplified by PCR using primers that are specific for the converted DNA but do not contain any CpG Cs in their sequences, and a probe which is specific for methylated DNA (M probe) or unmethylated DNA (U probe).

Nested BSP: DNA is first treated with sodium bisulfite to convert cytosine to uridine while leaving 5mC unchanged. The converted DNA is then amplified by two rounds of PCR using primers that are specific for the converted DNA but do not contain any CpG Cs in their sequences. The resulted PCR product is sequenced directly or after cloning.

CORBRA: DNA is first treated with sodium bisulfite to convert cytosine to uridine while leaving 5mC unchanged. The converted DNA is then amplified by PCR using primers that are specific for the converted DNA but do not contain any CpG Cs in their sequences. The resulted PCR product is digested with an appropriate restriction enzyme.

MSP: DNA is first treated with sodium bisulfite to convert cytosine to uridine while leaving 5mC unchanged. The converted DNA is then amplified by PCR using two pairs of primers, with one pair specific for methylated DNA (M pair) and the other for unmethylated DNA (U pair).

Methylight MSP: DNA is first treated with sodium bisulfite to convert cytosine to uridine while leaving 5mC unchanged. The converted DNA is then amplified by PCR using two pairs of primers, with one pair specific for methylated DNA (M pair) and the other for unmethylated DNA (U pair). In each of the PCR reactions, a probe is used to provide amplification signal.

Nested MSP: DNA is first treated with sodium bisulfite to convert cytosine to uridine while leaving 5mC unchanged. The converted DNA is first amplified by PCR using a pair of primers (outer primers) which is specific for converted DNA without discriminating methylated vs. unmethylated DNA. The resulted product is then re-amplified using two pairs of primers, with one pair specific for methylated DNA (M pair) and the other for unmethylated DNA (U pair). Options for primer design:

Degeneracy: For BSP, if CpG Cs are unavoidable in a primer sequence, two primers will be designed with the CpG Cs in the primers being degenerative, i.e., using letter C/T (Y) in forward primer, and G/A (R) in reverse primer.

Design primers on the minus strand: Primers will be designed on the minus strand of the input sequence.

Blat genome: If a genome is selected, you will be able to perform an in silico PCR to find potential mispairing of a pair of primers in the selected genome (which has been virtually bisulfite converted).

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, product size is usually smaller than that for regular PCR.

Primer size: For bisulfite PCR, 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 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 (BSP only): 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 CpG C at a primer’s 3’ end, e.g., a value of "3" means that a CpG C must appear at any of the last 3 bases of the 3' end.

CpG Cs in primer: The minimum number of CpG Cs 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 (M and U pair). This is useful only if you want to perform MSP using the same cycling conditions.

parameters for CORBRA

Choose an Enzyme: The restriction enzyme used to digest the resulted PCR product.

Min. enzyme cut: Minimal number of cuts in the product by the selected enzyme.

Terminus distance: The least distance allowed from the cut site to the nearest terminus of the PCR product.

Methylight

Probe size: The size of the probe.

Probe GC%: GC% of the probe.

3’G+C count: minimal number of G plus c at the 3' end of probe.

Tm higher: The difference of probe Tm over primer Tm.

Non CpG C: The number of non CpG Cs in probe.

CpG in probe (for BSP only): The number of CpG Cs in probe.

Nested BSP or MSP

Nested pair size: Product size for the outer primers.

primer info

Primer name: name of the primer.

Sequence: The sequence of the primer.

Len: Length of the primer.

Start: The start location of the primer in input sequence.

End: The end location of the primer in input sequence.

Degenerate: Whether degenerate primers are designed, if yes, Y for C/T and R for G/A.

CpGs: CpG Cs in primer.

C’s: Non CpG Cs in primer.

Self any: Primer self complementarity.

Self end: Primer 3' self complementarity.

Tm: The melting temperatures (Celsius) of the primer.

GC%: GC percentage of the primer.

Stability: Primer 3' stability

Score: Sum of quality scores.

Pair info:

Product size: PCR product size.

Pair start: PCR product start location in input sequence.

Pair end: PCR product end location in input sequence.

Comp any: Complementarily between primer pair.

Comp end: 3’ end complementarily between primer pair.

Tm: PCR product Tm.

Tm diff: Tm difference between primers.

Score: Sum score of primer pair.

Specificity: Check mispairing in selected bisulfite-converted genome.

Gene Info:

Predesigned BSP Primers: Primers are picked from the -500 to +200 region relative to TSS.

Predesigned MSP Primers: Primers are picked from the -500 to +200 region relative to TSS.