Differential display..... - (Jan/15/2008 )
Hi
I was trying to better understand differential display, I found the
protoco but I really cannot understand how
it is possible to amplify the cDNAs using "arbitrary" primers, I also
read that they are decamers(10nucleotides??), so did they actually put
all the possible combination of 10nucleotides?? I think that would be
statistically challenging, so I really do not understand how this
works, and also how can random primers anneal at the beginning of the
mRNAs and not just in the middle, thus amplifying just part of the
mRNA and deleting the part before, to me this really seems like a
mess.
Cheers!
-jonkye-
QUOTE (jonkye @ Jan 15 2008, 05:57 AM)
Hi
I was trying to better understand differential display, I found the
protoco but I really cannot understand how
it is possible to amplify the cDNAs using "arbitrary" primers, I also
read that they are decamers(10nucleotides??), so did they actually put
all the possible combination of 10nucleotides?? I think that would be
statistically challenging, so I really do not understand how this
works, and also how can random primers anneal at the beginning of the
mRNAs and not just in the middle, thus amplifying just part of the
mRNA and deleting the part before, to me this really seems like a
mess.
Cheers!
I was trying to better understand differential display, I found the
protoco but I really cannot understand how
it is possible to amplify the cDNAs using "arbitrary" primers, I also
read that they are decamers(10nucleotides??), so did they actually put
all the possible combination of 10nucleotides?? I think that would be
statistically challenging, so I really do not understand how this
works, and also how can random primers anneal at the beginning of the
mRNAs and not just in the middle, thus amplifying just part of the
mRNA and deleting the part before, to me this really seems like a
mess.
Cheers!
for what I remember when I did it 6 years ago, there are different sets of arbitrary primers that are designed to amplify a broad range of products, they are not representing all the possible permutations of 10 bp, otherwise you will have too many products, different sets of primers will amplify different "groups" of mRNA. And yes you will obtain only partial sequence, generally few hundreds of bp (average of 300-400 if i remember correctly), to identify the full sequence you will have to compare to databases or do 3' and 5' RACE.
good luck!
-cmonet-