Adding flanking regions to a gene for primer design
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3.0 years ago
eli_bayat ▴ 90

Hi, I am designing primers for pcr using primer3. the amplicon range should be between 1kb-2kb, however some of my genes that I am designing primers for have length < 1kb. As a result, the amplicon length will be < 1kb. I was suggested to add flanking regions manually to both side of the genes to get to the length > 1kb and then design the primers. I have few questions related to this:

Is adding flanking region to both sides of the gene and then design primer a way to go? If yes, is there a tool that given gene, it adds flanking region of length of interest to both side of the gene? or is there any other approach to do this?

Any suggestion appreciated.

Thanks

primer_design flanking amplicon primer3 flanking_region • 1.1k views
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I used to design my primers using the gene sequence available on ensembl. Just make sure to select the correct transcript for your purposes. Also, it's nice to use Gene Runner to 'test ' your primers together with Primer3 and UCSC.

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Thanks for your reply. I checked ensembl, and it seems it is for vertebrate genomes (unless I am not able to find other genomes) . I need to design primers for bacteria genes. Some of these genes have length smaller than 1kb, so not sure how to add flanking region to these genes to get to at least 1kb length and then design primers such that the amplicon size will be > 1kb. I checked if primer 3 has an option for flanking size or something similar and I couldn't find any. I also have multiple genes to design primers for, so I need a tool to be able to scale.

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