NDLI: Polymerase chain reaction-mediated gene synthesis: synthesis of a gene coding for isozyme c of horseradish peroxidase.

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Polymerase chain reaction-mediated gene synthesis: synthesis of a gene coding for isozyme c of horseradish peroxidase.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Shah, J. Fingar, S. A. Jayaraman, K. Fyles, J.
Description	Author Affiliation: Jayaraman K ( Life Sciences Division-Research Laboratories, Eastman Kodak Company, Rochester, NY 14650-2118.);
Abstract	The synthesis of a gene coding for horseradish peroxidase (HRP, isozyme c; EC 1.11.1.7) is described using a polymerase chain reaction (PCR)-mediated gene synthesis approach developed in our laboratory. In this approach, all the oligonucleotides making up the gene are ligated in a single step by using the two outer oligonucleotides as PCR primers and the crude ligation mixture as the target. The PCR facilitates synthesis and purification of the gene simultaneously. The gene for HRP was synthesized by ligating all 40 oligonucleotides in a single step followed by PCR amplification. The gene was also synthesized from its fragments by using an overlap extension method similar to the procedure as described [Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. (1989) Gene 77, 61-68]. A method for combining different DNA fragments, in-frame, by using the PCR was also developed and used to synthesize the HRP gene from its gene fragments. This method is applicable to the synthesis of even larger genes and to combine any DNA fragments in-frame. After the synthesis, preliminary characterization of the HRP gene was also carried out by the PCR to confirm the arrangement of oligonucleotides in the gene. This was done by carrying out the PCR with several sets of primers along the gene and comparing the product sizes with the expected sizes. The gene and the fragments generated by PCR were cloned in Escherichia coli and the sequence was confirmed by manual and automated DNA sequencing.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	10
Volume Number	88
Language	English
Publisher	National Academy of Sciences
Publisher Date	1991-06-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	DNA Biosynthesis Genes, Synthetic Horseradish Peroxidase Genetics Isoenzymes Polymerase Chain Reaction Chromatography, Gel Chromatography, High Pressure Liquid Cloning, Molecular Codon DNA Restriction Enzymes Escherichia Coli Molecular Sequence Data Oligonucleotides Metabolism Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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