Gene copy number determination - Gene copy number determination (Feb/03/2005 )
Gene copy number determination
Hi, I need to determine the gene copy number for a set of isoforms. I know that Southern blot is a technique commonly used for this but I have some concerns about the probe to use and result interpretation. Most people probe genomic DNA digested with three to four restriction enzymes with cDNA probes. But, without really knowing gene structure, this could give misleading results if one of the enzymes cuts in the intron region, giving two bands while there should be only one.
So far I have isolated three partial gene sequences and cloned three full length cDNAs which correspond with each partial gene sequences. I was planning to run a Southern blot with probes specific to one of the introns of each isoform. And, then, run another “conserved” probe to see if there are more copies than expected.
I would really appreciate any feedback about this idea and also whether there is or not a better, more reliable way to check for gene copy number.
I'd say do FISH (fluorescent in situ hybridization) but that's just me.
you can do quantitative PCR but I have no experiencce with the technique.