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Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 12 No. 3, Issue of July 15, 2009
© 2009 by Pontificia Universidad Católica de Valparaíso -- Chile Received June 4, 2008 / Accepted January 23, 2009
DOI: 10.2225/vol12-issue3-fulltext-2
RESEARCH ARTICLE

PCR assembly of synthetic human erythropoietin gene

Yazmin Bustami
School of Biological Sciences
Universiti Sains Malaysia
11800 Penang, Malaysia

Ahmad Ramli Mohd Yahya
School of Biological Sciences
Universiti Sains Malaysia
11800 Penang, Malaysia

Tengku Sifzizul Tengku Muhammad
Department of Biological Sciences
Faculty of Science and Technology
Universiti Malaysia Terengganu
21030 Kuala Terengganu, Terengganu, Malaysia 

Alexander Chong Shu-Chien#
School of Biological Sciences
Universiti Sains Malaysia
11800 Penang, Malaysia

Amirul Al-Ashraf Abdullah#
School of Biological Sciences
Universiti Sains Malaysia
11800 Penang, Malaysia

Mohd Azizan Mohd Noor
Malaysian Institute of Chemical and Bioengineering Technology
Universiti Kuala Lumpur
Lot 1988 Bandar Vendor, Tabuh Naning
78000 Alor Gajah, Melaka, Malaysia 

Yahya Mat Arip*
School of Biological Sciences
Universiti Sains Malaysia
11800 Penang, Malaysia
Tel: 60 4 653 4013
Fax: 60 4 656 5125
E-mail: ymarip@usm.my

*Corresponding author

Financial support: R&D initiative grant of Malaysian Institute of Pharmaceuticals & Nutraceuticals (07-05-IFN-BPH001). Yazmin Bustami is supported by fellowship from Universiti Sains Malaysia.

Present address: #Malaysian Institute of Pharmaceuticals and Nutraceuticals, Ministry of Science, Technology and Innovation, SAINS@USM, 10 Persiaran Bukit Jambul, 11900, Pulau Pinang, Malaysia.

Keywords: cloning, erythropoietin, oligonucleotide assembly.

Abbreviations:

CHO: Chinese hamster ovary
EPO: erythropoietin
huEPO: human erythropoietin
rhuEPO: recombinant human erythropoietin
PCR: polymerase chain reaction

Abstract   Full Text

Human erythropoietin (huEPO) is a glycoprotein with important physiological functions, such as erythropoiesis, angiogenesis, and wound healing. A therapeutic protein, huEPO is commonly used to treat patients suffering from renal and non-renal anemia. Recombinant human erythropoietin (rhuEPO) and endogenous huEPO are similar with respect to their biological and chemical properties. In this study, we describe the construction of synthetic huEPO gene to produce rhuEPO. The synthetic huEPO gene was constructed by overlapping oligonucleotides assembly and amplified by polymerase chain reaction (PCR). Twenty oligonucleotide sets, covering the huEPO gene sequence and two newly introduced restriction enzyme sites, were pulled together and amplified using Pfu DNA polymerase to produce the expected DNA products with sizes of ~500bp and ~600bp. The PCR products were ligated into pGEM-T plasmid vector to facilitate DNA sequencing process of the constructed huEPO gene and downstream cloning manipulation. DNA sequence analysis showed correctly assembled oligonucleotide sets, representing the huEPO gene sequence albeit with minor base mutations. Hence, oligonucleotides assembly and PCR amplification provide a convenient and speedy method for the synthesis of huEPO gene without depending on mRNA isolation and reverse transcription or the need to have a genomic library.

Supported by UNESCO / MIRCEN network