inverse pcr - (Apr/03/2011 )

Hi all

I am all set to do inverse pcr for isolation of 5'utr for a gene but i am having a few doubts , plz help me solve them
1. The digestion of genomic dna needs to be done and the bands needs to be self ligated , but when i check the genomic dna on gel ,it gives a smear. i used hindiii enzyme. How am i going to know which bands to choose?

2. The primers that i have designed are from the sequence that i have obtained from c-dna, how can i be sure about the primers as they might be in 2 different exons or present at a single strech?

Please help me clear my doubts.

thanks in advance

deespike on Sun Apr 3 09:56:50 2011 said:

You don't pick a band. Your primers will amplify the DNA flanked by your known sequence.
In inverse PCR, the genomic DNA is cut. Then the gDNA is ligated at low concentration to circularised the DNA fragments. Then the circular DNA molecules are cut with restriction enzyme in your known sequence. This is followed by PCR with primers to your known sequence. This PCR will amplify linear DNA fragments flanked by your known sequence. The PCR products are then cloned into a TA vector (modifications to the PCR insert is required if you used a proof reading polymerase) and transformed into bacteria. Isolated the plasmids from the e coli colonies and being walking the plasmid by sequencing.

deespike on Sun Apr 3 09:56:50 2011 said:

Silly question given that you are doing inverse PCR but has the genome you are working on been sequenced?
Well...one certainly with build primers that are adjacent to each other in a tail to tail orientation, flanking a restriction site. The only thing that comes to my mind at the moment is to run two inverse PCR which use different sets of primers. then you can compare the sequences produced by the two experiments to fill any holes.

Wait a minute.... you are trying to sequence the 5'utr! . Just build you inverse primers after the ATG start codon.Any introns between the primers will be missed but you have no interest in the coding sequence, only the 5'utr. So no problem.

Thanks a lot for your suggestions. It helps.