What % identity is enough between probe and template? - (Aug/30/2007 )
does anyone know if a probe that shows 70% identity to DNA from another member of the same gene family would pick up several bands on a Northern or give faulty positives in a library screen? The probe shows 100% identity to the correct gene and is 350 bp long, but it also shows 70% identity over 210 bp to another gene. Will this be a problem do you think? Short stretches within this region is very high in identity, 30 bp out of 31 bp are identical in one part for instance.
Any input would be nice.
I think your probe will hybridize with the other members of the family at 70% identity. If you are looking for selectivity, you should design a much shorter (oligo, perhaps) probe which is maximally different across the genes you are trying to differentiate. You might be better off with a PCR reaction to test for the presence of the gene you care about. Primers with mismatched 3' ends will be quite selective.
I agree with Phage.
You could vary the stringency of your washes to make your probe hyb more specific if there is no alternative in the probe design. Several High stringency washes may work but I wouldn't be that optimistic.