need some advise about chromosomal sequense orientation

Question

[monamehr1]

Hi everyone

I apologize if this is a basic question but I am so confused.

I want to do PCR-sequencing for the human-gene which has some psuedogens with high sequence similarity (97%) with the original gene.and I have no choice except designing primers at the regions with 1 or 2 mismatches from the psedogenes...

so I'm not sure if the psuedogenes would amplify to some extent with the original fragment or not at the end

I noticed that the orientation of the original gene at GeneCards browser is:

minus strand (Start:    2,138,711 bp from pter     End:    2,185,899 bp from pter )

and some psuedogenes are in:

plus strand (Start:    15,029,565 bp from pter     End:   up to 16,471,364 bp from pter)

Does it mean that my forward and reverse primers from the original gene can't amplify these psuedogenes?

thanks

mehr

Answer 1

[Devon Ryan]

No, it doesn't mean that. Strand has nothing to do with whether a primer will amplify a sequence. Just blast your primer sequences to see if they're likely to amplify the pseudogenes. If you're lucky, the pseudogene amplicon would either be a different length or have/not have a restriction enzyme site internally, which you could use for quick and easy quality control.

[monamehr1]

thanks Devon