Introduction Bisulfite Sequencing Methylation-Specific PCR

Target settings

Genome database UCSC - Homo sapien - hg38 - Genome (bisulfite-converted) UCSC - Homo sapien - hg19 - Genome (bisulfite-converted)

Targets in BED format (<= 100?, example, clear, SNP, Gene how-to)

Chromosome name examples chrM 1000 1001 chrM 2000 2001 chrM 3000 3001

Validation method Bisulfite Sequencing Methylation-Specific PCR Primers for bisulfite sequencing will avoid CG dinucleotides, while those for methylation-specific PCR will require a minimum number of CG sites in the 3' end region.

No-CG region at 3' end

bp

Length (from the primer's 3' end) where CG dinucleotides are not allowed.

Maximum allowed CG count

Max

Maximum number of CG dinucleotides allowed in the primer sequence.

3' end region for CG requirement

bp

Length of the region (from the primer's 3' end) where a minimum number of CG sites must be present.

Minimum CG sites in 3' region

Min

Minimum number of CG sites required within the defined 3' end region.

Quality control settings

Non-specific Amplicon T_m

Min

°C

The minimum binding stability between primer and its binding sites.

Non-specific Amplicon Size (bp)

From

To

Product size: 0 (min) - 1000000 (max).

Number of Off-targets

Min

Max

Number of non-specific amplicons.

Dimer ΔG

Min

kcal/mol

Primers with dimer stability exceeding this threshold will be discarded.

Hairpin Score (base pairs)

Min

Primers with hairpin stability exceeding this threshold will be discarded.

Minimum Nucleotide Differences

Min

Minimum number of different bases between target amplicon and off-targets if allowed.

Primer settings

Primer Min Size

bp

Primer Opt Size

bp

Primer Max Size

bp

Primer Min GC

%

Primer Opt GC

%

Primer Max GC

%

Primer Min T_m

°C

Primer Opt T_m

°C

Primer Max T_m

°C

Product settings

Product Min GC

%

Product Opt GC

%

Product Max GC

%

Product Min Size

bp

扩增子长度不能超过 5000 bp / maximum 5000 bp

Product Max Size

bp

扩增子长度上限 5000 bp（2026-06-04 起生效）。Maximum amplicon length: 5000 bp (effective 2026-06-04).

扩增子长度不能超过 5000 bp / maximum 5000 bp

Thermodynamic settings

Concentration of monovalent cations (usually KCl)

mM

Concentration of divalent cations (usually MgCl₂)

mM

Concentration of dNTPs

mM

Annealing oligo concentration

nM

Run