Can PCR be used to nonspecifically applify cDNA's?
Peter M. Muriana
muriana at aclcb.purdue.edu
Sat Feb 8 19:30:36 EST 1992
In article <92039.124334IO00865 at MAINE.MAINE.EDU>, IO00865 at MAINE.MAINE.EDU writes:
>Is it possible to amplify cDNA's before cloning using nonspecific PCR?
>What I have in mind is making double stranded cDNA using a standard
>protocol and the blunt end ligating a specific linker to each end
>of the DNA. PCR could then be performed using primers homologous to
>the linkers. This would (hopefully) give you nonspecific amplification.
>I think I've seen something like this in this newsgroup.
Yes, I solicited a week ago if non-specific PCR would be feasable
on a small amount of mixed probe of unknown sequence. I offered
something similar to random-primer PCR.
One response (my only one) suggested ligating a linker-adapter
to the ends, and then obtaining primers specific to the adapter
to run the PCR.
I also thought of using Terminal
Transferase for homopolymer tailing with dATP for instance, to
add a string of A's at the end of each molecule. Then use a
poly-dT primer (hexamer?) to run the PCR inward. Looking in a
molecular biology reference book, terminal transferase is suppose
to add 10-40 nucleotides, on average.
>If not is this likely to work on theoretical
>grounds or will you just get loads of short stuff amplified?
I guess that depends on the processivity of Taq polymerase?
>Thanks in advance
>IO00865 at maine RR#1 Box333
>Dept Plant biology and Pathology Old Town Maine, 04468
>Univ. Maine Orono 04469 207-394-2014
Hope this helps,
Name: Peter M. Muriana
Internet: muriana at aclcb.purdue.edu
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