Designing primers and hybridization probes - (Sep/07/2006 )
My research involves using both PCR and Fluorescent in situ hybridization (FISH) to detect wastewater pathogens. I've designed several primer pairs for the PCR reaction that have proven to be pretty specific. Could I use the same chromosomal (non-rRNA-coding) sequences to design FISH probes for the same species, instead of designing 16S probes as is the common practice in FISH? Why isn't this more commonly done in FISH research (I have found almost no references that have used the same DNA sequences for designing both primers and probes)? Do 16S probes have an advantage over non-16S probes? Could it be due to probe accessibility, i.e. 16S sequences on the chromosome are more accessible to hybridization probes? Any help would be greatly appreciated!
Yes, you could use the same chromosomal (non-rRNA-coding) sequences to design FISH probes, but you will have lower sensitivity campared to 16s one.
Why, because rDNA present in many many copy in nucleolus, which allow to have a more snesitive FISH.