search
for
 About Bioline  All Journals  Testimonials  Membership  News  Donations


Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 12, No. 3, 2009
Bioline Code: ej09031
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 12, No. 3, 2009

 en PCR assembly of synthetic human erythropoietin gene
Bustami, Y.; Yahya, A.R.M.; Muhammad, T.S.T.; Chien, A.C.S.; Abdullah, A.A-A; Noor, M.A.M. & Arip, Y.M.

Abstract

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.

Keywords
cloning, erythropoietin, oligonucleotide assembly

 
© Copyright 2009 - Pontificia Universidad Católica de Valparaíso -- Chile
Alternative site location: http://www.ejbiotechnology.info

Home Faq Resources Email Bioline
© Bioline International, 1989 - 2017, Site last up-dated on 16-Oct-2017.
Site created and maintained by the Reference Center on Environmental Information, CRIA, Brazil
System hosted by the Internet Data Center of Rede Nacional de Ensino e Pesquisa, RNP, Brazil