Nuclease protection assay - (Nov/17/2005 )
By using NPA, how to map 5' and 3' termini of mRNA?i
And how to determine introne-exon structure of the gene?
I don't need protocols for this method,because my lab doesn't do it, it is for my exam. I would be grateful if someone wrote me a theoretical explanation about it.
Milica
And how to determine introne-exon structure of the gene?
I don't need protocols for this method,because my lab doesn't do it, it is for my exam. I would be grateful if someone wrote me a theoretical explanation about it.
Milica
http://www.ambion.com/techlib/basics/npa/index.html
check out the above link.it might help.
u can use an antisense DNA probe which is labelled at the 5'-end for mapping the 5'-end
this probe shud be close to the start site ..after hybridization it can be treated with nuclease which cleaves off unhybridized regions..running an undigested sample and digested sample on the gel will tell u the difference in size.. using two such probes differing in target region..can confirm the start site
similarly u can use an antisense DNA probe which is labelled at the 3'-end for mapping the 3'-end
this probe shud be close to the start site ..after hybridization it can be treated with nuclease which cleaves off unhybridized regions..running an undigested sample and digested sample on the gel will tell u the difference in size.. using two such probes differing in target region..can confirm the start site
similarly u can use an antisense DNA probe which is labelled at the 3'-end for mapping the 3'-end
Thanks a lot!