PCR assembly of synthetic human erythropoietin gene

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 end...

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Autores principales:	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
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2009
Materias:	cloning erythropoietin oligonucleotide assembly
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000300011
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spelling	oai:scielo:S0717-345820090003000112010-04-01PCR assembly of synthetic human erythropoietin geneBustami,YazminYahya,Ahmad Ramli MohdMuhammad,Tengku Sifzizul TengkuShu-Chien,Alexander ChongAbdullah,Amirul Al-AshrafNoor,Mohd Azizan MohdArip,Yahya Mat 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.12 n.3 20092009-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000300011en10.4067/S0717-34582009000300011
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	cloning erythropoietin oligonucleotide assembly
spellingShingle	cloning erythropoietin oligonucleotide assembly 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 PCR assembly of synthetic human erythropoietin gene
description	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.
author	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
author_facet	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
author_sort	Bustami,Yazmin
title	PCR assembly of synthetic human erythropoietin gene
title_short	PCR assembly of synthetic human erythropoietin gene
title_full	PCR assembly of synthetic human erythropoietin gene
title_fullStr	PCR assembly of synthetic human erythropoietin gene
title_full_unstemmed	PCR assembly of synthetic human erythropoietin gene
title_sort	pcr assembly of synthetic human erythropoietin gene
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2009
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000300011
work_keys_str_mv	AT bustamiyazmin pcrassemblyofsynthetichumanerythropoietingene AT yahyaahmadramlimohd pcrassemblyofsynthetichumanerythropoietingene AT muhammadtengkusifzizultengku pcrassemblyofsynthetichumanerythropoietingene AT shuchienalexanderchong pcrassemblyofsynthetichumanerythropoietingene AT abdullahamirulalashraf pcrassemblyofsynthetichumanerythropoietingene AT noormohdazizanmohd pcrassemblyofsynthetichumanerythropoietingene AT aripyahyamat pcrassemblyofsynthetichumanerythropoietingene
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PCR assembly of synthetic human erythropoietin gene

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