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PCR assembly of synthetic human erythropoietin gene
Bustami, Yazmin; Yahya, Ahmad Ramli Mohd; Muhammad, Tengku Sifzizul Tengku; Shu-Chien, Alexander Chong; Abdullah, Amirul Al-Ashraf; Noor, Mohd Azizan Mohd; Arip, Yahya Mat.
  • Bustami, Yazmin; Universiti Sains Malaysia. School of Biological Sciences. Penang. MY
  • Yahya, Ahmad Ramli Mohd; Universiti Sains Malaysia. School of Biological Sciences. Penang. MY
  • Muhammad, Tengku Sifzizul Tengku; Universiti Malaysia Terengganu. Faculty of Science and Technology. Department of Biological Sciences. Kuala Terengganu. MY
  • Shu-Chien, Alexander Chong; Universiti Sains Malaysia. School of Biological Sciences. Penang. MY
  • Abdullah, Amirul Al-Ashraf; Universiti Sains Malaysia. School of Biological Sciences. Penang. MY
  • Noor, Mohd Azizan Mohd; Universiti Kuala Lumpur. Malaysian Institute of Chemical and Bioengineering Technology. Alor Gajah. MY
  • Arip, Yahya Mat; Universiti Sains Malaysia. School of Biological Sciences. Penang. MY
Electron. j. biotechnol ; 12(3): 11-12, July 2009. ilus, tab
Article Dans Anglais | LILACS | ID: lil-551889
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.
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Texte intégral: Disponible Indice: LILAS (Amériques) Sujet Principal: Érythropoïétine / Clonage d'organisme Limites du sujet: Humains langue: Anglais Texte intégral: Electron. j. biotechnol Thème du journal: Biotechnologie Année: 2009 Type: Article / descriptif de projet Pays d'affiliation: Malaisie Institution/Pays d'affiliation: Universiti Kuala Lumpur/MY / Universiti Malaysia Terengganu/MY / Universiti Sains Malaysia/MY

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Texte intégral: Disponible Indice: LILAS (Amériques) Sujet Principal: Érythropoïétine / Clonage d'organisme Limites du sujet: Humains langue: Anglais Texte intégral: Electron. j. biotechnol Thème du journal: Biotechnologie Année: 2009 Type: Article / descriptif de projet Pays d'affiliation: Malaisie Institution/Pays d'affiliation: Universiti Kuala Lumpur/MY / Universiti Malaysia Terengganu/MY / Universiti Sains Malaysia/MY