Specific Amplification of Difficult PCR Products from Small
Amounts of DNA Using FastStart Taq DNA Polymerase
The availability of a specific, clean polymerase chain
reaction (PCR) product in sufficient amounts prior to
cycle sequencing is one important prerequisite for the
generation of a good-quality DNA sequence. Since heterozygote
samples must be unambiguously identified,
excellent DNA quality is particularly crucial for applications
such as mutation detection or discovery of single
nucleotide polymorphisms (SNPs).
As the PCR efficiency is controlled by many parameters –
such as polymerase type, buffer type, primer concentration
and stability, dNTP purity and concentration, cycling
parameters, as well as complexity and concentration of
starting template – it is not possible to establish one
standard PCR amplification protocol. Therefore, it is necessary
to optimize PCR conditions for each PCR amplicon.
Successful amplification of difficult templates (e.g.,
templates containing GC-rich regions) often requires the
addition of cosolvents (e.g., dimethylsulfoxid [DMSO]) to
lower the DNA-strand separation temperature. However,
this method has the drawback of inhibiting enzyme activity
and slowing down the extension rate – which is a
problem if only a small amount of DNA is available.
Therefore, we have compared the performance of Taq
DNA Polymerase and FastStart Taq DNA Polymerase in
PCR amplifications of difficult (GC-rich) DNA templates
with different starting amounts of DNA.
Materials and Methods
A 540-bp DNA fragment from 5-hydroxytryptamine
receptor 2C (HTR2C) with a GC-content of 65 % was
amplified in a total volume of 50 µl using Taq DNA
Polymerase or FastStart Taq DNA Polymerase on a thermal
cycler (MJ Research Tetrad, Watertown, MA).
20 ng of human genomic DNA was amplified using
0.2 mM of each primer (5’-CAGCCATCCGGGACCTGTC-3’ and 5’-ACCTGCCGATTGCATATGAAC-3’) in the presence
of the buffers supplied with the enzyme (10mM Tris
pH8.3, 50 mM KCl, 1.5mM MgCl2 for Taq Polymerase;
50mM Tris pH 8.3, 10 mM KCl, 2.0mM MgCl2, 5mM
[NH]2SO4 for FastStart Taq DNA Polymerase), 200 mM of
dNTPs, and 1.5U enzyme. The concentration of DMSO
or GC-rich solution (provided with FastStart Taq DNA
Polymerase) ranged from 0 –10 %.
The thermocycling protocol consists of an initial incubation
at 95 °C for 15 minutes followed by 35 cycles at 94 °C
for 1 minute, 60 °C for 30 seconds, 72 °C for 1minute, and
a final extension step of 72 °C for 10 minutes.
The resulting 540-bp PCR products were electrophoresed
through a 1.5% agarose gel containing
ethidiumbromide, then visualized under UV light.
Results and Discussion
It was not possible to obtain the desired 540 bp amplicon
in sufficient amounts without the addition of DMSO or
GC-rich solution using Taq DNA Polymerase or FastStart
Taq DNA Polymerase.
The addition of DMSO and GC-rich solution improved
the reactions with respect to specificity and yield
(Figure 1). 2% DMSO or 2 –10 % GC-rich solution resulted
in the amplification of a specific PCR product using
both enzymes, but with a higher yield using FastStart
Taq DNA Polymerase. Therefore, FastStart Taq – in contrast
to Taq Polymerase – does not react to DMSO or
GC-rich solution by slowing down its extension rate.
DMSO in concentrations higher than 2% inhibits the
activity of Taq Polymerase, but does not inhibit the activity
of FastStart Taq DNA Polymerase. FastStart Taq DNA
Polymerase, in combination with GC-rich solution, which
has been especially developed and recommended by
Roche Applied Science, seems to be most suitable for templates with an even higher GC-content of 65 %, without
any inhibition of enzyme activity.
To determine the sensitivity of both enzymes, dilutions of
human genomic DNA (50 pg, 100 pg, 200 pg, 500 pg, 1 ng,
2 ng, 5 ng, 10 ng, 20 ng) were amplified in the presence of
2% DMSO or 2% GC-rich solution (Figure 2). The threshold
of detection is defined as the amount of DNA (present
in the dilution) that shows the PCR signal of the 540-bp
fragment on the gel (see arrows). Taq DNA Polymerase
amplified a visible PCR product in 2% DMSO reactions
with ≥10 ng template DNA, and in 2% GC-rich solution
reaction with ≥2 ng of DNA. FastStart Taq DNA
Polymerase showed at least 10 times higher sensitivity,
generating visible PCR products in reactions with only
100 pg of DNA template. These results are in agreement
with the results obtained with different concentrations of
cosolvents: The higher sensitivity of FastStart Taq DNA
Polymerase can be explained by its robustness in the presence
of additives (e.g., DMSO or GC-rich solutions).
FastStart Taq DNA Polymerase did not seem to be inhibited
by these cosolvents and did not slow its extension rate.
Summary
We have successfully used FastStart Taq DNA Polymerase
to obtain clean and specific PCR products from GC-rich
regions of small amounts of template DNA. In addition to
the advantage of hot start for specificity, FastStart Taq
Polymerase is not inhibited even by high amounts of
cosolvents, such as DMSO or GC-rich solutions, and provides
clean PCR products for DNA sequencing from
DNA-template amounts as little as 100 pg. Therefore it is
superior to Taq DNA Polymerase for this application.