PROOF-READING DNA POLYMERASE
Highly accurate and Easy-to-use
is a new, highly thermostable, high performance proofreading DNA
polymerase developed and manufactured by Qbiogene Molecular
Biology in collaboration with Ifremer. Originally isolated from
Pyrococcus abyssi, this enzyme was discovered to possess close
homology with the enzyme from Pyrococcus furiosus (Pfu) and
display a similar high fidelity in template copying. Its robust
DNA polymerase and 3 to 5 exonuclease activities,
combined with its higher thermostability, provide a superior
choice for accurate and efficient DNA amplification whilst
outperforming other popular proof-reading DNA polymerases.
To test the flexibility of this enzyme for DNA cloning purposes,
parallel PCR reactions were carried out using Isis, Pfu and Taq
DNA polymerases. The amplified product was a 1.82 Kb
kanamycin-resistant mini-transposon, provided on a plasmid
template. Since copy errors are rare with Isis, we intentionally
chose non-optimal amplification conditions to increase the
chances of PCR misincorporation. Each PCR contained excess
template (50 ng) and primers (100 pmol: universal and reverse)
along with a high concentration of dNTPs (200M). Nevertheless,
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 94C,
30 sec at 52C and 3 min at 72C. At the end of the program,
the tubes were placed on ice prior to analysis of 5 l by gel
electrophoresis on a 1% agarose gel.
1. Amplification of a 1.82 Kb kanamycin-resistant mini-transposon
using Taq, Pfu and Isis DNA polymerases.
these conditions, we observed that Taq DNA polymerase
reproducibly outperformed the proof-reading polymerases in terms
of the absolute quantity of DNA product, whilst Isis in turn
provided more product than the cloned Pfu polymerase (Fig 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).
Clone products efficiently
The PCR reactions from above were purified from unused primers
using a GENECLEAN Turbo for PCR (Bio101 1103-200). The products
were then digested with EcoRI and SpeI (Qbiogene Molecular
Biology EREC4200, ERSPE200, buffer III) and cloned into the
EcoRI-XbaI sites of pUC18. Parallel ligations were performed for
2 h at 37C, using similar concentrations of each PCR fragment,
after repurification of the digested fragments by RPM Turbo.
Each ligation was then transformed into chemically competent
Escherichia coli DH10B cells before plating on LB agar
(containing 100 g ml-1 ampicillin and 50 g ml-1 kanamycin).
After an overnight incubation at 37C, the number of colonies
obtained was 350, 250 and 200 for the Taq, Isis and Pfu DNA
polymerases respectively. Plasmid minipreps were performed using
the RPM Turbo Kit (Bio101 2066-200). Out of 6 mini-preps each,
6 contained the correct insert for Isis, whilst Taq and Pfu
scored 5 and 3 respectively.
unwanted PCR errors
The cloned inserts were sequenced using the universal and reverse
sequencing primers by the dideoxy chain termination method
(Qbiogene ResearchServices, SQMS0001). The sequences were
compared to the original template by alignment using the BLAST
engine (Blast 2.0: http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html). The
results are shown in Table 1.
1: Sequence errors incorporated into PCR products
increase the statistical significance of these results further,
sequence data is currently being obtained and will be added
information : visit Isis DNA Polymerase page