BLAST for sequences that are more distantly related

[peerah]

Hi all,

Is there a way to set up NCBI online BLAST to look for sequences that are more distantly related to a subject sequence and have them show up within the top 100? I tried tweaking some settings I saw but seemed to only get very close matches. I am designing a PCR to amplify a family of proteins that is pretty well conserved, but would like to make sure it doesn't amplify something that is outside of acceptable homology.

Thank you!

[diego diaz]

If you are blasting amino acids, you have to change the substitution matrix, from blosum62 to blosum45, to align distantly related proteins.

[kmcarr]

PSI-BLAST was designed exactly for this purpose. PSI==Position Specific Iterative. It is an iterative search which first consturcts a Position Specific Scoring Matrix (PSSM) from a multiple sequence alignment of related proteins. That PSSM is used to further seach a database (instead of the standard BLOSUM or PAM matrices). The search can be repeated iteratively to refine the PSSM and pull in more distantly related proteins.

Read more here.