• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. May 15, 1991; 88(10): 4084–4088.
PMCID: PMC51602

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

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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Hoyle MC. High resolution of peroxidase-indoleacetic Acid oxidase isoenzymes from horseradish by isoelectric focusing. Plant Physiol. 1977 Nov;60(5):787–793. [PMC free article] [PubMed]
  • Welinder KG. Covalent structure of the glycoprotein horseradish peroxidase (EC 1.11.1.7). FEBS Lett. 1976 Dec 15;72(1):19–23. [PubMed]
  • Welinder KG. Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase. Eur J Biochem. 1985 Sep 16;151(3):497–504. [PubMed]
  • Jayaraman K, Shah J, Fyles J. PCR mediated gene synthesis. Nucleic Acids Res. 1989 Jun 12;17(11):4403–4403. [PMC free article] [PubMed]
  • Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. [PMC free article] [PubMed]
  • Zhang H, Scholl R, Browse J, Somerville C. Double stranded DNA sequencing as a choice for DNA sequencing. Nucleic Acids Res. 1988 Feb 11;16(3):1220–1220. [PMC free article] [PubMed]
  • Springer BA, Sligar SG. High-level expression of sperm whale myoglobin in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8961–8965. [PMC free article] [PubMed]
  • Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. [PubMed]
  • Keohavong P, Thilly WG. Fidelity of DNA polymerases in DNA amplification. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9253–9257. [PMC free article] [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Tindall KR, Kunkel TA. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry. 1988 Aug 9;27(16):6008–6013. [PubMed]
  • Eckert KA, Kunkel TA. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990 Jul 11;18(13):3739–3744. [PMC free article] [PubMed]
  • Fujiyama K, Takemura H, Shibayama S, Kobayashi K, Choi JK, Shinmyo A, Takano M, Yamada Y, Okada H. Structure of the horseradish peroxidase isozyme C genes. Eur J Biochem. 1988 May 2;173(3):681–687. [PubMed]
  • Smith AT, Santama N, Dacey S, Edwards M, Bray RC, Thorneley RN, Burke JF. Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme. J Biol Chem. 1990 Aug 5;265(22):13335–13343. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

    Your browsing activity is empty.

    Activity recording is turned off.

    Turn recording back on

    See more...