Biology Forum › Molecular Biology › LT-PCR!!!
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- May 3, 2006 at 7:59 am #4666
2810712ParticipantLT-PCR[ Ligated Templet-PCR]
Now primer designing separately for each isoform is a bit… taking time.
So what if we do the following-
1. ligate a known DNA sequence with the original
2. Then send a primer for that known sequence & have ur PCR done then! U need just one common primer!
nOW, HOW TO GET THE ORIGINAL & PRIMER SEQUENCES SEPARATED AGAIN… if u,atall , need it ; because u know the ligated DNA’s sequence & molecular wt. & by correct placement of stop & start codogenes on the ligated new DNA, u can stop the expression of the new DNA too, so u may not require their separation.I’ve less practical exposure so please convey ur opinions…
Any ideas…
hrushikesh
- May 3, 2006 at 1:20 pm #478062810712Participant
Self Ligation-PCR
If we ligate the separated strands from same ds DNA & then add some non-ligated ssDNA from the source, u can have a PCR , i think.
This will omit the problem of separation of the ligated new DNA in the LT-PCR.DP-PCR [ degradation primer -PCR]
Degrade some of the source DNA & then add it to the SL-DNAs which will give mixture of diff. lengths of the replicated DNA…But all these require a bit more source DNA isn’t it?
SP-PCR[ self-primer PCR]
One more possibility is to cut the source DNA with an endonuclease, then ligate it with the original complete source DNA, then the secondary structures formed will make it appear dsDNA at some parts, where the replication can begin.
& why don’t we use natural mechanism of primase action to create primers..?
Have a share of ur ideas too, waiting curiously.
hrushikesh
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