In silico PCR tool

Virtual (in silico) or electronic PCR (ePCR) primers/probes or microRNA (miRNA) sequences or guide RNA (gRNA) binding at closely matched sequences with a focus on eliminating "off-target" effects against a whole genome (chromosome) prediction of likely PCR products and search for potential mismatches of the specified primers or probes. Search for multiple targets simultaneously within a specified range. In silico PCR primer searching is useful for the discovery of melting temperature target-binding sites.

An alternative command line Java application in silico PCR for local use without restrictions on genome size and the number of files to be analyzed.

Input format: sequence can be pasted or uploaded as a file in FASTA format or retrieved sequence (NCBI’s accession, e.g. A02710) from NCBI’s "nuccore" nucleotide database.
Size Limitations: the length of the query sequence and the size of the batch file are theoretically unlimited.
Degenerate primer sequences are also accepted, each letter represents a combination of one or several nucleotides: B=(C,G,T), D=(A,G,T), H=(A,C,T), K=(G,T), M=(A,C), N=(A,C,G,T), R=(A,G), S=(G,C), V=(A,C,G), W=(A,T), Y=(C,T). U=Uracil, I=Inosine and LNA: dA=E, dC=F, dG=J, dT=L.

Upload or paste sequence(s) in FASTA format:

Amplicon detection:		C>>T bisulfite conversion
Minimal amplicon size (bp):		Show all matching sites of primer binding
Maximal amplicon size (bp):		Show amplicon sequences
Mismatches allowed at the 3'-terminus:		Show only amplicons lengths
Generate		Clear

To export the results: select all (Ctrl-A), copy (Ctrl-C) and paste (Ctrl-V).

С >> T bisulfite conversion (bisulfite modified genome)

Sequence, design of specific PCR primers for in silico bisulphite conversion for both strands - only cytosines not followed by guanidine (CpG methylation) will be replaced by thymines:

5’aaCGaagtCCCCa-3'        5’aaCGaagtTTTTa-3'
  |||||||||||||     ->      ||||||:|::::| 
3’ttGCttCaggggt-5'        3’ttGCttTaggggt