Long PCR advice needed
johnw86684 at aol.com
Wed May 17 08:36:11 EST 1995
I have used Perkin Elmer's XL-PCR kit for amplification of not only DNA,
but also fairly long (up to 7 kb) RNA. Given that the difficulty of
amplifying long RNA is greater than that of amplifying DNA, some of my
observations are probably relevant. In general, the XL-PCR kit protocol
worked well, with some very slight modifications.
1. Design PCR primers to have Tm's which are a) higher than normal and b)
closely-matched. Ideally, both primers would have Tm's within 1 deg. of
68 deg. (using Oligo, for example).
2. Additionally, I observed considerably more robust amplification when
adding a 10-15 nt non-complementary 5'-extension (generally containing
useful sequences, such as cloning sites, etc.) onto the primers. This
resulted in 40-50-mer primers, but much more amplicon was obtained with
the extended primers. This observation held true under my PCR conditions,
for several primer sets. I believe that primers whose Tm is predicted to
be 68 +/- 1 deg., a 5'-extension of 12 nt on each primer, and an
anneal/extend temperature of 68 deg. will work well, in most cases.
3. Use the kit's recommended cycling conditions. For reproducible
results, a high-performance cycler such as Perkin Elmer's 9600 is
recommended. I did obtain amplification using other cyclers and
rationally modified cycling profiles, but the 9600 was almost always
4. My work with long RT-PCR involved partially-unknown viral sequences.
The success of long PCR seems to be quite strongly dependent on having
close match between primers and template. This is not unexpected, given
the high anneal/extend temperatures which are used.
5. In the same way that the first few cycles of standard PCR are critical
for establishing the specificity of amplification, the same is true for
long PCR. Troubleshooting in some cases may necessitate adjusting upward
the anneal/extend temperature for the first 3-5 cycles, or in other ways
optimizing only those first several cycles.
6. One important difference between RT-PCR and DNA PCR will be in the
total amount of DNA per reaction. The XL-PCR kit may mention this, but
it's worth noting here that lowering the amount of total DNA may improve
the specificity of the reaction. In the beginning, for a single copy
target for example, one might want to run several dilutions of the sample,
ranging from 10 ng to 1 ug.
Hope this is helpful. I think you'll find the protocols booklet that
comes with the XL-PCR kit well-written and useful.
JohnW86684 at aol.com
More information about the Methods