The application is based on FastPCR software and provides professional facilities for designing primers for most PCR applications: standard, inverse, long-distance, real-time (TaqMan and MGB-probe assay design), and bisulfite PCR primer design.
Input Format: Sequence(s) can be pasted or uploaded as a file in FASTA format.
Primer design: | ||
Minimal length (nt): | ||
Maximal length (nt): | ||
Minimal Tm (°C): | ||
Maximal Tm (°C): | ||
Minimal Linguistic Complexity (%): | ||
All possible combinations of '[ Forward ]' with '[ Reverse ]' within the sequence(s):
1. [ ]
2. [ ] [ ]
3. [ [ ] ]
Oligonucleotide specificity is one of the most critical factors for good PCR; optimal primers should hybridize only to the target sequence, especially when using complex genomic DNA as a template. Amplification problems can occur when primers anneal to repetitive sequences (retrotransposons, transposons or inverted tandem repeats). Alternative product amplification can also happen when primers are complementary to inverted repeats and produce multiple bands. However, the generation of inverted repeat sequences is exploited in two common generic DNA fingerprinting methods (RAPD).
Hydrolysis probes are typically labelled with a fluorophore reporter at the 5′end and a quencher [such as the Black Hole Quencher (BHQ)] at the 3′end. During amplification, the DNA polymerase exonuclease activity cleaves off the reporter, allowing for signal unquenching and detection. Hydrolysis probes can be amended for increased binding stability or signal specificity. For example, MGBs selectively bind non-covalently to the minor groove, a shallow furrow in the DNA helix. Dual-labelled probes conjugated with MGB groups form extremely stable duplexes with single-stranded DNA targets, enabling shorter probe lengths and superior quenching.