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Polymerase-endonuclease amplification reaction for large-scale enzymatic production of antisense oligonucleotide

Xiaolong Wang¹ and Deming Gou²

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1. Ocean University of China, Department of Biotechnology
2. University of Illinois at Chicago, Pediatrics

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Document Type:

Manuscript

Date:

Received 02 September 2009 04:00 UTC; Posted 02 September 2009

Subjects:

Biotechnology, Chemistry, Pharmacology

Tags:

• miRNA
• thermostable DNA polymerase
• thermostable endonuclease
• DNA amplification
• Antisense oligonucleotide

Abstract:

Synthetic oligonucleotides are contaminated with highly homologous failure sequences. Oligonucleotide synthesis is difficult to scale up because it requires expensive equipments, hazardous chemicals, and tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves >100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation, so it has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

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This document is licensed to the public under the Creative Commons Attribution 3.0 License

How to cite this document:

Wang, Xiaolong and Gou, Deming. Polymerase-endonuclease amplification reaction for large-scale enzymatic production of antisense oligonucleotide. Available from Nature Precedings <http://hdl.handle.net/10101/npre.2009.3711.1> (2009)

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