19. How should I select a set of primers to use
for PCR?
See: Innis, M. A., and D. H. Gelfand. 1990. Optimization of PCRs.
In:
PCR Protocols: A guide to methods and applications. Academic
Press,
New York.
Here are some general pointers:
1. Try to keep the primer 50% G-C give or take 15%. If overly G-C
rich add
a string of As or Ts at 5' end; If overly A-T rich, do the same
with Gs and Ts.
2. Try to avoid Gs and Cs at 3' end of the primers. This may
increase
the chance of forming primer dimers.
3. Avoid self-annealing regions within each primer.
4. Compute Tm as sum of 4 C for G/C and 2 for A/T, then subtract
5 C
from this value and that is our annealing temp. Naturally, the
annealing
temp will be that of the primer with the lower value. Differences
of 4-6 C
do not seem to affect yield of PCR. Ideally you would like the Tm
for each
primer to match and be within the 70-75 degrees C range.
5. A good practice is to check the target DNA sequence if it is
known
for mispriming areas. A quick check scanning the sequence of
vector for
approximately 70% and above homolgy regions can help prevent
obtaining
multiple contaminating bands in your PCR.
21. What is "Hot-start" PCR?
"Hot-start" PCR is a method that generally produces
cleaner PCR products.
Template DNA and primers are mixed together and held at a
temperature above the
threshold of non-specific binding of primer to template. All the
PCR reaction
components are added for the extention reaction except one
critical reagent
(usually the thermostable polymerase).
Just prior to the cycling, the missing component is added to
allow the reaction
to take place at higher temperature. Due to lack of non-specific
hybridization
of primers to template, the amplified DNA bands tend to be
cleaner; the primers
don't have a chance to anneal non-specifically.
This method is difficult to do because the tubes must be kept on
a 100C heat
block as your work surface. There are ways to avoid this however.
One way is to
quickly cool the tubes on ice while adding the component mix. You
can then heat
the tubes on the pre-warmed thermocycler just before adding the
last component.
This may not always be successful due to a thermal ramp that may
allow
non-specific interactions between primer and template.
Hot starts are also done by creating a physical barrier between
the essential
components, eg. primers and template. This barrier may be created
by putting a
half-reaction mixture into the bottom of the tube and melting wax
over the mix.
The wax used can be "PCR Gems" from Perkin-Elmer/Cetus
or any number of
home-grown waxes (e.g. paraffin or Paraplast). Cooling solidifies
the wax, and
the missing components can be placed on top. The mixing of the
last component
then occurs at high temperature only when the wax melts and the
top half-mix is
added by convection currents within the tubes. The PCR then
proceeds as a
normal cycle sequence.
Co-solvents have also been used to eliminate artifacts from PCR
reactions. For
high fidelity, the specificity of primer to template is
desirable. Co-solvents
such as glycerol, DMSO, and formamide, work to provide highly
stringent
reactions by changing the Tm of the primer-template hybridization
reaction.
Co-solvents have various effects on the thermostablility of the
polymerase
enzyme. Glycerol tends to extend the resistance of Taq enzyme to
heat
destruction, while formamide lowers enzyme resistance.
In some cases, it may be necessary to add single-strand DNA
binding protein in
order to keep DNA with a high GC content from forming secondary
structures.
This may also be a problem in cycle sequencing reactions.
See the following references for more details:
Dutton, C. M., C. Paynton, and S. S. Sommer. 1993. General method
for
amplifying regions of very high G + C content. Nucleic Acids
Research
21:2953-2954.
Blanchard, M.M., Taillon-Miller, P., Nowotny, P., Nowotny, V.
1993. PCR buffer
optimization with uniform temperature regimen to facilitate
automation. PCR
Methods and Applications 2: 234-240.
Rapley, R., S. Flora, and M. R. Walker. 1992. Direct PCR
sequencing of murine
immunoglobulin genes using E. coli single-stranded DNA-binding
protein. PCR
Methods and Applications 2:99-101.
Chou, Q. 1992. Minimizing deletion mutagenesis artifact during
Taq DNA
polymerase PCR by E.coli SSB. Nucleic Acids Research 20:4371.
Wainwright, L. A., and H. S. Seifert. 1993. Paraffin beads can
replace mineral
oil as an evaporation barrier in PCR. BioTechniques 14:34-36.
Horton, R. M., Hoppe, B. L., and B. M. Conti-Tronconi. 1994.
AmpliGrease:
``hot start'' PCR using petroleum jelly. BioTechniques 16:42-43.
D'Aquila, R. T., Bechtel, L. J., Videler, J. A., Eron, J. J.,
Gorczyca, P.,
and J. C. Kaplan. 1991. Maximizing sensitivity and specificity of
PCR by
preamplification heating. Nucleic Acids Research 19:3749.
Chou, Q., Russell, M., Birch, D. E., Raymond, J., and W. Bloch.
1992.
Prevention of pre-PCR mis-priming and primer dimerization
improves
low-copy-number amplifications. Nucleic Acids Research
20:1717-1723.