Very basic RT-PCR question

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Fri May 18 03:11:25 EST 2001


In article <9e0j3g$gmm$1 at uni00nw.unity.ncsu.edu>, the eminent Karthik
Aghoram at North Carolina State University wrote
>I am almost embarassed to ask this, but here I go anyhow:  Are there any 
>known limits to the range of reverse transcriptase?  One of my colleagues 
>is using a long-range thermo-polymerase to amplify long sequences and 
>build a library, but if the RT "fell off" before reaching the end of the 
>CDS, one is s**ewed, right?  

I'm sure I've seen a paper in Analytical Biochemistry which RT'd a 20kb
or more template. They had to use an RNaseH minus RT.

As to the other post suggesting lowering the temp. I would have said no
to that. In a long template there is bound to be some sort of secondary
structure that the enzyme cannot handle and  so it stops. Raising the
temperature, without killing the RT, could melt that secondary
structure, giving you longer cDNA.

There are an increasing no. of higher temp RT's around i.e. AMV RNAseH
minus which will work at 60C. Maybe try a mix of that with MMULV RT
RNaseH minus at say 50-55C. MMULV Rt will not like the higher temps for
too long.

Duncan 
-- 
Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR: 

Optimisation, Probe Technology & Future Systems

4-5 September 2001
King Alfred's College, Winchester, UK 

http://www.dera.gov.uk/html/news/events/real_time_quantitative_pcr.htm




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