Comparison of primer design and oligonucleotide analysing tools versus other most popular on-line primer design and analysing packages


Features FastPCR NCBI/Primer-BLAST (Primer3) BatchPrimer3 (Primer3) IDT SciTools: PrimerQuest, OligoAnalyzer 3.1 PerlPrimer BiSearch Web server Primo Pro 3.4
Specificity
Primer or probe design, length (nt) 12-500 15-30   16-35 12-30 10-35 13-36
Limit for sequence length (nt) no limit 50,000   no limit no limit 5000 no limit
Relative calculation speed very quick slow slow slow slow very slow quick
Enable high-throughput runs, multiple templates (sequences or primers) and multiple targets inside each sequence yes no yes no no no yes
Individual PCR options for sequence yes yes yes yes yes yes yes
Degenerated nucleotides at all operation (Tm calculation, searches and probe, primer design etc.) yes no yes yes no yes no
LNA and other nucleotide modifications yes no no yes no no no
Primer PCR efficiency and linguistic complexity determination: the average value for the linguistics complexity (LC) of primers yes,
LC=91.1±3.6% (6000 primers)		 no,
LC=79.6±9.4% (6000 primers)		 no no,
LC=84.4±8.1% (407 primers)		 no no,
LC=73.2±10.8% (524 primers)		 no
Calculation of optimal annealing temperature yes no no no no no no
Primer dimers, self-dimer and for alternative to Watson-Crick base pairing, like wobble base pairs or G/C-quadruplexes detection
Primer's 3'-end cross and self-dimers yes Errors yes yes yes yes yes
Primer's internal cross and self-dimers yes Errors no Errors yes yes no
No Watson-Crick base pairing detection yes no no no no no no
Alternative amplification
BLAST search no yes no no yes yes yes
internal sequence test yes yes no no no Errors Errors
external (specific library) test yes yes no no yes yes no
PCR applications
Multiplex PCR with pair primers or (and) single primer(s) yes no no no no no no
in silico PCR for multiple sequences and primers, and searching multiple targets simultaneously within a certain range yes no no no no partial no
Universal and unique PCR yes no no no no no no
Inverted PCR and circular sequence yes no no no no no no
Bisulphite modification PCR assays and in silico PCR yes no no no no yes no
Polymerase Extension PCR multi-fragments assembly cloning yes no no no no no no
Design Tiling Arrays/PCR yes no no no no no no
Loop-mediated Isothermal Amplification (LAMP) yes no no no no no no
Polymerase Chain Assembly (PCA) yes no no no no no no
Provides graphical web interface yes yes yes yes yes yes yes
Primer design parameters validated in lab yes yes yes yes yes yes yes
Overall assessment (0-10 points) 9+ 6 4 5 3 3 2