How to eliminate gDNA from RT-PCR?
ladasky at leland.Stanford.EDU
Tue Jul 22 13:42:47 EST 1997
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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