Experience with NASBA

Bob van Gemen bvgemen at am.otbc01.umc.akzonobel.nl
Mon Nov 10 10:36:27 EST 1997


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Dear colleagues,

It is interesting to notice the recent discussion on the NASBA (or 3SR)
nucleic
acid amplification technology. We have been working with the technology
extensively and maybe the marginality as observed by comparing the
number of
NASBA publications versus the number of PCR publications does not say
anything
about the true quality of the technology.

Actually NASBA does what every nucleic acid amplification technology
does: it
lifts the RNA (or DNA) amount from only a few copies to easily
detectable
amounts (10e13 to 10e14 copies). And, interestingly, NASBA does so in an

isothermal manner. Like any other technology in this field NASBA has its
strong
points that make it better than other technologies FOR CERTAIN
APPLICATIONS.

Obviously, NASBA is very good in amplifying RNA and as result we have
been very
successful in using NASBA for qualitative and quantitative detection of
RNA
(both genomic and messenger RNA), most recently even in a multiplex
format.
Another obvious application for an isothermal nucleic acid amplification

technology is in situ nucleic acid amplification. Some problems have
been encountere when using intact cells, but on tissue sections the
technology has been relatively successfull.

Recently we have used NASBA together with some molecular beacons
("folded
probes") and accomplished real time signal measurement in a closed tube.
This
new methodology can be of value in high throughput systems without the
fear for
contamination. And another obvious item: the isothermal nature of NASBA
will
facilitate the automation process of the method.

These are just a few points were NASBA has the edge over the other
amplification
technologies out there, like PCR. Maybe it is the familiarity of the
research
community with PCR that make us all a little hesitant in trying
alternative
amplification technologies. My experience is, however, that those who
dared to
take the chance of trying something new were rewarded with nice results.

Maybe also a few words concerning the problems one may encounter with
NASBA.
These are not much different from any problems one would see with
RT-PCR. In our
hands contamination is the biggest worry. And for that there two
solutions:
closed tube format (see above) or if nothing  else an anti-contamination
method.
For that we have some experience with a method that works as well as the

dUTP/UNG method for PCR but it will never give a 100% protection. It is
better
(and easier) to have some strict lab rules and clean up actions from
time to
time.

Bob van Gemen
Organon Teknika
R&D, biotechnology research unit



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<TT></TT>

<P><TT>Dear colleagues,</TT><TT></TT>

<P><TT>It is interesting to notice the recent discussion on the NASBA (or
3SR) nucleic</TT>
<BR><TT>acid amplification technology. We have been working with the technology</TT>
<BR><TT>extensively and maybe the marginality as observed by comparing
the number of</TT>
<BR><TT>NASBA publications versus the number of PCR publications does not
say anything</TT>
<BR><TT>about the true quality of the technology.</TT><TT></TT>

<P><TT>Actually NASBA does what every nucleic acid amplification technology
does: it</TT>
<BR><TT>lifts the RNA (or DNA) amount from only a few copies to easily
detectable</TT>
<BR><TT>amounts (10e13 to 10e14 copies). And, interestingly, NASBA does
so in an</TT>
<BR><TT>isothermal manner. Like any other technology in this field NASBA
has its strong</TT>
<BR><TT>points that make it better than other technologies FOR CERTAIN
APPLICATIONS.</TT><TT></TT>

<P><TT>Obviously, NASBA is very good in amplifying RNA and as result we
have been very</TT>
<BR><TT>successful in using NASBA for qualitative and quantitative detection
of RNA</TT>
<BR><TT>(both genomic and messenger RNA), most recently even in a multiplex
format.</TT>
<BR><TT>Another obvious application for an isothermal nucleic acid amplification</TT>
<BR><TT>technology is in situ nucleic acid amplification. Some problems
have been encountere when using intact cells, but on tissue sections the
technology has been relatively successfull.</TT><TT></TT>

<P><TT>Recently we have used NASBA together with some molecular beacons
("folded</TT>
<BR><TT>probes") and accomplished real time signal measurement in a closed
tube. This</TT>
<BR><TT>new methodology can be of value in high throughput systems without
the fear for</TT>
<BR><TT>contamination. And another obvious item: the isothermal nature
of NASBA will</TT>
<BR><TT>facilitate the automation process of the method.</TT><TT></TT>

<P><TT>These are just a few points were NASBA has the edge over the other
amplification</TT>
<BR><TT>technologies out there, like PCR. Maybe it is the familiarity of
the research</TT>
<BR><TT>community with PCR that make us all a little hesitant in trying
alternative</TT>
<BR><TT>amplification technologies. My experience is, however, that those
who dared to</TT>
<BR><TT>take the chance of trying something new were rewarded with nice
results.</TT><TT></TT>

<P><TT>Maybe also a few words concerning the problems one may encounter
with NASBA.</TT>
<BR><TT>These are not much different from any problems one would see with
RT-PCR. In our</TT>
<BR><TT>hands contamination is the biggest worry. And for that there two
solutions:</TT>
<BR><TT>closed tube format (see above) or if nothing&nbsp; else an anti-contamination
method.</TT>
<BR><TT>For that we have some experience with a method that works as well
as the</TT>
<BR><TT>dUTP/UNG method for PCR but it will never give a 100% protection.
It is better</TT>
<BR><TT>(and easier) to have some strict lab rules and clean up actions
from time to</TT>
<BR><TT>time.</TT><TT></TT>

<P><TT>Bob van Gemen</TT>
<BR><TT>Organon Teknika</TT>
<BR><TT>R&amp;D, biotechnology research unit</TT>
<BR><TT></TT>&nbsp;
<BR><TT></TT>&nbsp;</HTML>

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