Primer Specificity

Question

[Surendra]

how can i determine primer (upto 10bp) specificity in genome(prokaryotic and eukaryotic)?

[Michael Schubert]

Did you already take a look at http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi?

Answer 1

[Sean Davis]

For genomes available via the UCSC genome browser, you can use the In Silico PCR tool.

Answer 2

[Pierre Lindenbaum]

Did you try Primer-Blast?

Primer-BLAST was developed at NCBI to help users make primers that are specific to the input PCR template. It uses Primer3 to design PCR primers and then submits them to BLAST search against user-selected database. The blast results are then automatically analyzed to avoid primer pairs (all combinations including forward-reverse primer pair, forward-forward as well as reverse-reverse pairs) that can cause amplification of targets other than the input template.

Answer 3

[Wubin Qu]

Try:

http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/

or,

http://biocompute.bmi.ac.cn/MFEprimer/

[Wubin Qu]

Please use the new server: http://www.mfeprimer.com/

Answer 4

[Felix Francis]

Try ThermoAlign: A primer design tool for tiled amplicon sequencing (includes multiplexing) using highly specific primers.

Manuscript: http://www.nature.com/articles/srep44437

https://github.com/drmaize/ThermoAlign