need help...how to compare mouse vs human sequences for primer design - (Jan/08/2009 )

hey everyone,

i have stably transfected a 3t3 cell line (mouse cell line) with plasmids carrying a gene that i am looking to overexpress. i need to check via pcr to ensure my gene is in fact in the cells. what i am looking to do is design a set of primers to amplify a portion of my gene (a human gene).

problem i am running into is that there is decent homology between the human and mouse gene homologs so if i pick any random primer set, i am likely to be amplifying the native gene (mouse) in the cell line in addition to my overexpressed human gene. i am trying to get around this by picking primers from areas where there is less homology (specifically where there are base pair differences in the primers' 3' end).

what i would like to do now is use a tool to see if the primers ive picked will amplify the mouse gene. is there a way to do this with BLAST? (input the mouse gene, input my primers (which are from the human gene) and see if it will amplify to any extent).

any help would be greatly appreciated. im pretty new to this so other strategies would be great too.

Do an alignment of the mouse and human sequences on an alignment program or webpage (ClustalW is good) and then anchor primers in non-conserved regions, if there are any.

Easy peasy. Enter the sequence of your primers into nucleotide BLAST and BLAST against Mouse genomic + transcript.

http://blast.ncbi.nlm.nih.gov/Blast.cgi

If you have an exact hit you'll know that your primers aren't going to distinguish between the two genes.

Would it be possible to have one primer annealing in the promoter region of your transfected DNA?