cDNA - (Apr/10/2008 )
When cDNA is constructed in the RNA is uracel, does this uracel also base pair with the "A" or not,
Uracel have ability to make double bond with the A nucleotide or not.
This RNAse H will cleave the single stranded RNA becasue it its uracel identification
why this DNA nuclease just cleave the hair pin loop.
Hi,
I don't know if this helps 'cause your post confused me a little bit.
cDNA is single stranded, just like RNA. You have to use a DNA polymerase to make it double stranded so I don't understand what is the pairing problem.
Can you make your problem more specific?
I don't quite get what is your question.
When make cDNA I though we always use 'A', 'T', 'C', 'G', thus there are not suppose to have 'U' in cDNA.
Yes if RNA form double strand and pair together, 'U' is pair with 'A', U-A pair is formed by hydrogen bond (yes 2 of them).
As I understand, no RNase can touch double strand RNA (perfect match no bouble or loop).
So, when RNA secondary structure hair pin form, only single strand loop will be eaten up by RNase but not double strand stem.
That is what I remember, and hope it helps
when you obtain cDNA it is double stranded, not single stranded (the "c" stands for complementary). you use mRNA to get cDNA. in vivo uracil bonds with adenine (RNA doesn't use thymine). if i'm not mistaken you're using a cDNA kit which provides a RNAse H, the idea of using it is to get rid of any RNA after you've synthesized the cDNA, not to destroy your template RNA.
I mean when your cDNA is prepared and it is prepraed from the template RNA,
cDNA is the in the first strand and now its is attached with the template RNA, this template RNA is degraded by RNAse H because of its uracel identity.
now you have to create second strand of the cDNA.....
end of the cDNA make a some hair pin loop structure becasue of self priming and you can use DNA polymerase to make the secd DNA,
After making this second strand you can destroy the stem loop at the end of double strandes by DNAse, does i ma right
IN THE RNA there will be uracel and it can be recognised by the RNAse H and so it will cleave the RNA template and i think the Hair pin lool is degraded by single strand DNA nuclease
cDNA is the in the first strand and now its is attached with the template RNA, this template RNA is degraded by RNAse H because of its uracel identity.
now you have to create second strand of the cDNA.....
end of the cDNA make a some hair pin loop structure becasue of self priming and you can use DNA polymerase to make the secd DNA,
After making this second strand you can destroy the stem loop at the end of double strandes by DNAse, does i ma right
Hi,
I didn't make myself clear the last time. When I said cDNA was single stranded I meant you synthesize first strand cDNA. If you want do make second strand cDNA you have to remove the RNA template using RNase, making a single stranded cDNA molecule, and then produce the second strand using a DNA polymerase. Did I make myself any clearer now?