Question: How To Find A 28Bp 'Primer' Sequence In A Genome?
gravatar for Dan
10.0 years ago by
Dan510 wrote:

Dumb question I know, but...

What tool to use?

I'd like to find forward and reverse primers within an approximate distance of each other... I'd like to accept 1 or two mismatches. The genome is ~1 Gbp. I have ~500 forward reverse pairs to find in the genome.

Any hints appreciated :-)

genome sequence search tool primer • 6.8k views
ADD COMMENTlink modified 5.3 years ago by Timothée Flutre20 • written 10.0 years ago by Dan510

I think you need to be more specific with your question: what are you trying to do? 500 pairs of "primers": are you targeting something in particular or just random? Will these sequences be used for wet lab experiments like PCR or qPCR? Etc.

ADD REPLYlink written 10.0 years ago by Nicojo1.1k

do you need some random primers or do you know your 500 targets ?

ADD REPLYlink written 10.0 years ago by Pierre Lindenbaum126k

Yes, please be more clear. Do you (1) have 500 forward and reverse 28 bp sequences that you wish to align to the genome sequence? Or (2) want to generate 500 forward/reverse primers using the genome sequence?

ADD REPLYlink written 10.0 years ago by Neilfws48k
gravatar for Darked89
10.0 years ago by
Barcelona, Spain
Darked894.2k wrote:

Did not used it myself yet, but there is a new program claiming to be better than primer3:


Download it from:

ADD COMMENTlink written 10.0 years ago by Darked894.2k

thanks for the links, didn't know about that.

ADD REPLYlink written 10.0 years ago by brentp23k
gravatar for Michael Dondrup
10.0 years ago by
Bergen, Norway
Michael Dondrup47k wrote:

I know that many people use the primer3 software to design primers. There are also two web-interfaces where you can try out the parameters, and there are many of them. The program eprimer3 is part of the EMBOSS tools, maybe the best way of getting a command-line executable of primer3.

I didn't see a mismatch parameter though, because for a 'real' PCR-primer to allow for mismatches is a bad idea. I noted the '' in your question, so you might not really be looking for primers. So what is it then? If this is just a piece of sequence then you do not need to bother with uniqueness or melting temperature constraints.

ADD COMMENTlink written 10.0 years ago by Michael Dondrup47k
gravatar for brentp
10.0 years ago by
Salt Lake City, UT
brentp23k wrote:

[EDIT] Just saw this today ( which could be another piece of software to check out. I didnt read it carefully, but it looks like it builds on primer3. [/EDIT]

Since you have so few pairs, you could just run them through a read-aligner allowing mismatches, and find pairs that are aligned within X basepairs in the reference genome. Actually, you could probably even use the paired-end feature of most aligners so that the aligner only find the pairs with nearby matches. For either of those methods, you could use bowtie.

As far as I know, primer3 does allow gaps and mismatches, so that may also be an option if you just want to see if you have good primers, but you'd probably have to use the command-line version.

or, you could use BLAST and find nearby hits between the pairs.

ADD COMMENTlink modified 9.9 years ago • written 10.0 years ago by brentp23k
gravatar for Madelaine Gogol
9.9 years ago by
Madelaine Gogol5.1k
Kansas City
Madelaine Gogol5.1k wrote:

You could try e-PCR or in-silico PCR. e-PCR is available to run locally.

ADD COMMENTlink written 9.9 years ago by Madelaine Gogol5.1k

So can isPCR, Source code can be found here:

ADD REPLYlink written 9.2 years ago by Casey Bergman18k
gravatar for Timothée Flutre
5.3 years ago by
Timothée Flutre20 wrote:

(better late than never)

One can use blastn with -task blast-short, and it's quite fast. As explained in the manual (section BLAST+ features), "blastn-short is BLASTN program optimized for sequences shorter than 50 bases". Further down (table C2), it says "blastn-short is optimized for sequences less than 30 nucleotides" and specifies word_size=7 (versus 11 for blastn) and reward=1 (versus 2 for blastn), all the other parameters remaining as for blastn.

In a Unix-like machine, with NCBI BLAST 2.2.26+:

zcat genome.fa.gz | formatdb -i stdin -l formatdb.log -p F -n genome


blastn -query primers.fa -db genome -task blastn-short -out primers_one_genome.txt -outfmt 7

If one knows both primers around the sequence they amplify, I recommend concatenating them into a single sequence, and map it to the genome. BLASTn-short will usually (likely?) return two best hits, one per primer, separated by a gap on the genome.

ADD COMMENTlink modified 17 months ago by RamRS25k • written 5.3 years ago by Timothée Flutre20
gravatar for Wubin Qu
8.6 years ago by
Wubin Qu160
Wubin Qu160 wrote:

MFEprimer has an command-line to check the specificity of primers, you can download it from the web site:

ADD COMMENTlink written 8.6 years ago by Wubin Qu160

Please use the new server:

ADD REPLYlink written 2.6 years ago by Wubin Qu160
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