RT-PCR of highly coiled RNA

Tim Fitzwater TFITZWATER at somalogic.COM
Tue Jan 29 14:23:51 EST 2002


1. Try adding betaine to 1 or 2 M final concentration.  The anhydrous or
monohydrate forms (from Sigma) will dissolve at 5.5 M.

AMV and HIV RT pauses are reduced in the presence of betaine.  (D. S.
Mytelka, 1995, Ph.D. thesis, University of California, Berkeley cited in D.
S. Mytelka and M/ C. Chamberlin 1996 Analysis and supression of DNA
polymerase pauses associated with a trinucleotide repeat.  Nucleic Acids
Res. 24 (14) 2774-2781.)
T. Weissensteiner reported that in his hands, betaine added to MMLV RT
reactions did not improve the yields or relative amplification efficiencies
of his templates as measured by PCR, but that MMLV RT was more active at
45°C and 50°C in the presence of betaine.(T. Weissensteiner, 
http://www.bio.net/hypermail/methods/current/01/1.html on March 8, 2000.) So
this effect may be template specific.

2. ThermoScript RT is available from Invitrogen.  ThermoScript RT can be
used at 50-65°C.  Invitrogen recommends incubation at 50-55°C for most
targets, but incubation at 50-60°C can be used for oligo(dT) and 50-65°C for
gene-specific primers in order to reduce secondary structure or to improve
specificity.  (If random hexamers are used, incubate at 25°C for 10 minutes
to extend the primers before incubating at 50-65°C.)  Terminate reaction by
heating at 85°C for 5 min.

3. C. therm (Carboxydothermus hydrogenoformans) Polymerase (Roche Applied
Science) for magnesium-dependent reverse transcription of GC-rich sequences
up to 4 kb at 60°C to 70°C. RT-PCR kit also available.
http://biochem.roche.com/pack-insert/2016311A.pdf.  5.	GC-rich RNA sequences
can be amplified by RT-PCR in the presence of 5-10% DMSO and 1-2 M betaine.

4. Enhanced AMV RT is available from Sigma and can also work at elevated
temperature.



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