NASBA, not what it is, but what problems one encounters

elader at ambion.com elader at ambion.com
Wed Nov 5 11:36:30 EST 1997


In article <632ucp$9h422 at ncisun1-nf0.ncifcrf.gov>,
  pnh at ncifcrf.gov (Paul N Hengen) wrote:
>
> Michael Vork (Michael.Vork at FYS.rulimburg.nl) wrote:
>
> > I am glad that everybody wants to show what he or she knows. I know
> > what NASBA is, that was not the question. I am looking for
> > information about this system from experienced NASBA users.
> > Does anybody has some real information ???
>
> I doubt that you'll make any friends that way. I had no idea what you
> were talking about, so I was the one who asked what NASBA is. Before
> throwing around any more undefined acronyms, I wanted to learn something
> for myself. Sorry to be ignorant...

In any case, I both know what NASBA is (nucleic acid sequence based
amplification) and have used it extensively for quite a while. BASBA, and
it's sister technique 3SR are isothermal, sequence specific, RNA
amplification systems.

In a nutshell: a pair of gene specific primers, one of which has a T7
promoter on it, AMV RT, T7 RNA polymerase, RNase H, dNTPs, NTPs, and an
RNA sample.

First strand synthesis of cDNA is initiated with the T7-GSP. Second
strand is made with the 5' primer and the RT (along with the RNaseH ala
gubler hoffman). Once the ds DNA is made, T7 makes many copies of one
strand as aRNA. The aRNA is templatre for more reverse transcription. All
this happens at 42 degrees and, if you are lucky, makes an amazing amount
of specific RNA from a total RNA sample in an hour or two.

Advantages over PCR - isothermal, only works off RNA (no problems with
genomic contamination), simple to perform once optimized, incredibly
sensitive

Disadvantages - more work to optimize (lots), isothermal cyclic rxns lead
to more mutations than pcr, strong bias for smaller products (also
because no discrete cycles)

Organon Technika has a quantitative HIV test out that uses NASBA.
Not alot of NASBA in USA it seems.

Cehck out review articles in Science a few years back. Compton is the
author I think.

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