Display Settings:

Format

Send to:

Choose Destination
J Biochem Biophys Methods. 2008 Apr 24;70(6):820-2. doi: 10.1016/j.jprot.2007.12.009. Epub 2008 Jan 9.

A modified method for PCR-directed gene synthesis from large number of overlapping oligodeoxyribonucleotides.

Author information

  • 1Neuroscience Discovery Research Wyeth Research, CN8000, Princeton, NJ 08543, USA.

Abstract

Here we report an improved, reproducible, simple, rapid, and cost-effective PCR-based DNA synthesis method using short (25-40 bp) overlapping oligodeoxyribonucleotides (oligos). The method involves two steps; (1) assembly of multiple/overlapping oligos by PCR to generate the template DNA and (2) amplification of the template DNA sequence with the two outermost oligos as primers. We have tested this method by synthesizing approximately 35 genes ranging in size between 300 bp and 1700 bp and G+C content from moderate (30%) to high (65%). In addition, we used the method to introduce 29 mutations simultaneously into a single gene. Key to the success of this method is the use of optimized oligo concentrations and the type of DNA polymerase used. This simplified and highly reproducible method is expected to be beneficial for the synthesis of a wide variety of genes.

PMID:
18272229
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for Elsevier Science
    Loading ...
    Write to the Help Desk