How to eliminate gDNA from RT-PCR?

John Ladasky ladasky at leland.Stanford.EDU
Tue Jul 22 13:42:47 EST 1997


Hi there,

	I designed an RT-PCR which gave me a product of the expected size
(1 kB).  I prepared my mRNA from a cell line using the Invitrogen Fast
Track mRNA Kit.  When I cloned and sequenced the RT-PCR products, I was
surprised to discover that all nine of my first-round clones were not
cDNA sequences, but instead were nearly identical to a recently-published
processed pseudogene sequence.  I had the same in-frame, premature stop
codons that were reported, so there's no doubt that I've amplified from
contaminating genomic DNA.  If I replace my 5' primer with one substan-
tially downstream, or if I try a different gene that does not have a par-
alogous processed pseudogene, I get cDNA sequences with no problem.

	I'm willing to bet that many of us are using mRNA preps that con-
tain contaminating genomic DNA.  However, because the genomic sequences are
usually longer owing to the presence of introns, it is easy to eliminate
them from RT-PCR.  Processed pseudogenes are nasty because they have no
introns, and thus they are the same length as the cDNA sequence.

	I am entertaining ideas about how I can eliminate the gDNA contam-
ination.  I would be grateful if you could comment on the following ideas.

	1) Use a different method of mRNA isolation.  Does anyone know
	   that the method of mRNA preparation that they use is particular-
	   ly good at removing gDNA?

	2) Treat the mRNA with DNAse.  A colleague in my lab has tried this
	   without success.  She thinks that most DNAse preps have contam-
	   inating RNAse activity (and I agree that this is likely).

	3) Treat the reverse-transcribed mRNA with DNAse.  Is this any dif-
	   ferent than approach 2?  What happens to DNA-RNA hybrids in the
	   presence of nucleases?

	4) Treat either the mRNA or the reverse-transcribed mRNA with a
	   restriction enzyme that cuts the pseudogene sequence in the ge-
	   nomic DNA, rendering it unusable as a template for PCR.  I have
	   used this approach successfully in order to eliminate side prod-
	   ucts in regular PCR.  But as with approaches 2) and 3), I am 
	   concerned about unwanted nuclease activity that might degrade 
	   mRNA or DNA-RNA hybrids.

-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology
Keywords  : immunology, music, running, Green



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