Finding non-polymorphic region of genome for PCR primer design
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24 months ago
joneill4x ▴ 110

I working with a highly heterozygous species. I want to find a region of the genome with no polymorphism.

I am looking to design PCR primers as a positive control for a presence/absence marker. The PCR primers should amplify a non-polymorphic area of genome. The amplicon size should be 50-75 bp.

I thought about using the BUSCO gene set, and placing the primers in the exon of a BUSCO gene. The exon of a BUSCO region is likely to be non-polymorphic, but I am worried about contamination of the reaction with DNA from another species. Since BUSCO genes are highly conserved across all species, the primers might amplify the DNA of another contaminating species.

Does anyone have any tips on finding a non-polymorphic area of the genome which can be used as a template for PCR primer design?

Thank you

PS - I don't have sequencing data for all the individuals which will be subject to this marker and it's positive control.

PCR genome polymorphism • 387 views
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The amplicon size should be 50-75 bp.

That is too short for a PCR reaction, considering a primer size of 21-25 bp, you are amplifying only 6-30 bp.

Does anyone have any tips on finding a non-polymorphic area of the genome which can be used as a template for PCR primer design?

If you have a full genome, you can first exclude all repetitive regions, so check if you have a repeat annotation with Repeat Masker/Modeler or similar tool.

You are also right to consider a gene as a possible non-polymorphic region, but focus on genes which any mutation is lethal, genes like histones, actin, basal transcription factors and other (incorrectly called "house-keeping" genes) are your main targets.

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