allele or two copies? - (Dec/02/2008 )
hi everyone!
i am pcring a gene with the genome DNA.i got two bands in the gel,one is A ,and the other is B.Then i pcred the same gene in different lines,and i found some lines only had band A,some lines had B,and some lines had two bands. i sequenced all the band,and i found A and B have a very high similarity in the exon,but A had a insertion in the intron.i want to kown the A and B are the allele in the same locus or they are two copies in the different loci?how to prove that?
Roger
i am pcring a gene with the genome DNA.i got two bands in the gel,one is A ,and the other is B.Then i pcred the same gene in different lines,and i found some lines only had band A,some lines had B,and some lines had two bands. i sequenced all the band,and i found A and B have a very high similarity in the exon,but A had a insertion in the intron.i want to kown the A and B are the allele in the same locus or they are two copies in the different loci?how to prove that?
Roger
A variation on Loss-of-native-allele realtime PCR assay can confirm this. Technically difficult to standardize.
There should be some time-tested standard method to address your problem, but I can't think of any.
A FISH experiment is my first thought. If you make a fluorescent probe to the intron, does it localize to the same chromosome as a fluorescent probe made to the exons? If it's in a different place, then you could guess that you are looking at two different loci. Another experiment you could maybe do is a southern blot. You know what the sequence of your gene is with or without the intron. Assuming that you know the sequence of some surrounding genes, then you could predict the sizes of different restriction products in your two samples (with or without the intron). Cut your genomic DNA with these different restriction enzymes and probe with the intron or exon. Do the sizes match your predictions, or are they completely different? If they are completely different then the surrounding DNA is different and you're probably looking at different loci.
You could also design primers from your known gene sequence and sequence the flanking parts to see whether they are at the same place or at another locus