PCR assembly of synthetic human erythropoietin gene
Ahmad Ramli Mohd Yahya
Tengku Sifzizul Tengku Muhammad
Amirul Al-Ashraf Abdullah#
Mohd Azizan Mohd Noor
Yahya Mat Arip*
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.
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.