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.

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: http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html).

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.

Isis DNA Polymerase™ product page

Related products:
PCR Polymerases

Reference:
(1). Gueguen et al. PCR performance of the highly thermostable proofreading B-type DNA polymerase from hyperthermophilic euryarchaeon Pyrococcus abyssi. (Manuscript in preparation).