PCR Primer Software Comparison

Comparison of primer design and oligonucleotide analysis tools versus other most popular online packages.

Yes Supported No Not supported Partial Limited Errors Known issues

Features	FastPCR	NCBI / Primer-BLAST	BatchPrimer3	IDT SciTools	PerlPrimer	BiSearch	Primo Pro 3.4
Specificity
Primer or probe design length (nt)	12–500	15–30	—	16–35	12–30	10–35	13–36
Sequence length limit (nt)	no limit	50 000	—	no limit	no limit	5 000	no limit
Relative calculation speed	very quick	slow	slow	slow	slow	very slow	quick
High-throughput: multiple templates & targets	yes	no	yes	no	no	no	yes
Individual PCR options per sequence	yes	yes	yes	yes	yes	yes	yes
Degenerate nucleotides (Tm, search, design)	yes	no	yes	yes	no	yes	no
LNA & other nucleotide modifications	yes	no	no	yes	no	no	no
PCR efficiency & linguistic complexity (LC)	yes LC = 91.1 ± 3.6 % (6 000 primers)	no LC = 79.6 ± 9.4 % (6 000 primers)	no	no LC = 84.4 ± 8.1 % (407 primers)	no	no LC = 73.2 ± 10.8 % (524 primers)	no
Optimal annealing temperature calculation	yes	no	no	no	no	no	no
Primer Dimers, Self-Dimers & Non-Watson-Crick Pairing
3′-end cross & self-dimers	yes	Errors	yes	yes	yes	yes	yes
Internal cross & self-dimers	yes	Errors	no	Errors	yes	yes	no
Non-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 (pair & single primers)	yes	no	no	no	no	no	no
In silico PCR — multiple sequences & primers	yes	no	no	no	no	partial	no
Universal & unique PCR	yes	no	no	no	no	no	no
Inverse PCR & circular sequences	yes	no	no	no	no	no	no
Bisulphite-modification PCR & in silico PCR	yes	no	no	no	no	yes	no
Polymerase Extension PCR multi-fragment assembly	yes	no	no	no	no	no	no
Tiling Arrays / PCR design	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
General
Graphical web interface	yes	yes	yes	yes	yes	yes	yes
Lab-validated primer design parameters	yes	yes	yes	yes	yes	yes	yes
Overall Assessment
Score (0–10)	9+	6	4	5	3	3	2

Feature data compiled from publicly available documentation and hands-on testing of each tool.