Application
note Experiment
to check yield, cloning efficiency and error rate of Isis DNA Polymerase™
To test the
performance of this enzyme for DNA cloning purposes, parallel PCR reactions
were carried out using Isis and Pfu DNA Pol (Taq DNA Pol
was included as a control). The amplified product was a 1.82 Kb kanamycin-resistant
mini-transposon, provided on a plasmid template. Identical PCR conditions
were used for each enzyme. No time was invested to optimize yield or fidelity
: each PCR contained excess template (50 ng) and primers (100 pmol: universal
and reverse) along with a high concentration of dNTPs (200µM) ;
each polymerase was used at the concentration and in the buffer recommended
by the manufacturer. The reactions were set up on ice immediately prior
to performing 30 PCR cycles of 1 min at 94°C, 30 sec at 52°C and
3 min at 72°C. At the end of the program, tubes were placed on ice
prior to analysis of 5 µl by gel electrophoresis on a 1% agarose
gel.
Yield
Under the above conditions, we observed that Isis DNA Polymerase™
provided more product than cloned Pfu DNA polymerase (Figure
1). Under similar conditions, no such difference was observed for
a 220 bp fragment, with each enzyme providing an essentially equivalent
high yield of product (data not shown).
Figure 1.
Amplification of a 1.82 Kb kanamycin-resistant mini-transposon using
Pfu and Isis DNA polymerase™.
Cloning
efficiency
The PCR products from above were purified and cloned into pUC18. Parallel
ligations were performed for 2 h at 37°C, using similar concentrations
of each PCR fragment, after repurification of the digested fragments.
Each ligation
was then transformed into chemically competent Escherichia coli DH10B
cells before plating on LB agar (containing ampicillin and kanamycin).
After an overnight incubation at 37°C, the number of colonies obtained
was 250 for Isis DNA Polymerase™ and 200 for Pfu DNA Pol (350
for Taq DNA Pol). To check cloning efficiency, 6 colonies from
each plate were picked at random followed by plasmid purification and
sequencing.
Isis
DNA Polymerase™
6 out
of 6 contained the correct insert
Pfu
DNA Polymerase
3 out
of 6 contained the correct insert
(Taq
DNA Polymerase
5 out
of 6 contained the correct insert)
Error
rate
The cloned inserts were sequenced using the universal and reverse sequencing
primers by the dideoxy chain termination method . The sequences were compared
to the original template by alignment using the BLAST engine
(Blast 2.0: ).
Table
1: Sequence errors incorporated into the 1.8 kb PCR products
DNA
Polymerase
Total
bases sequenced
Nucleotide
substitutions
Error
frequency per Kb
Isis
DNA Polymerase™
9196
(for 6 clones)
0
0.00
Pfu
DNA Polymerase
4313
(for 3 clones)
0
0.00
Taq
DNA Polymerase
6269
(for 5 clones)
6
0.95
No errors
were detected for Isis DNA polymerase™ over a total of 9196 bases
sequenced, despite the high concentrations of nucleotides used in these
experiments which are known to increase the chance of misincorporation.
Conclusion
Isis DNA Polymerase™ is ideally suited for cloning purposes where
DNA length and accurate template copying are requisite. Also, the enzyme
requires minimal optimization.
Reference:
(1). Gueguen et al. PCR performance of the highly thermostable proofreading
B-type DNA polymerase from hyperthermophilic euryarchaeon Pyrococcus abyssi.
(Manuscript in preparation).
