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

>Ethan Strauss
>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
   Phone: 317-494-8284



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