Nucleic Acids Research, 2000, Vol. 28, No. 2 E5-e5
© 2000 Oxford University Press
Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions
Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington, DC 20375-5348, USA
We have developed a novel biochemical method to simultaneously amplify and immobilize a target gene onto insoluble particles using PCR. This method employs the covalent attachment of one of two PCR primers to a particle surface either directly during DNA synthesis of the primer or post-DNA synthesis, through the use of chemical crosslinkers. Immobilization of the target gene can be achieved directly during PCR amplification, with one bead-bound primer and one soluble primer. Alternatively, this can be achieved post-PCR, through covalent attachment of a chemically modified primer incorporated into the amplicon to an activated particle. All of the immobilized DNA templates containing appropriate regulatory regions were fully competent for transcription and translation reactions and several could be re-used in serial reactions. The most successful strategy utilized amino-silanized controlled pore glass beads, which were coupled to phosphorylated primers using carbodiimide chemistry. These bead-bound primers were used during PCR to generate attached DNA templates that could be collected and re-used for at least seven sequential transcription reactions without significant loss in efficiency. This method has also been successfully applied to the amplification, transcription and translation of multiple DNA templates using a single, immobilized primer. The combined PCR-based amplification/immobilization method was shown to be more durable than post-PCR chemical immobilization and affords the convenience of performing sequential PCR amplification, transcription and translation reactions in a single tube.
* To whom correspondence should be addressed at: Naval Research Laboratory, Code 6950, Center for Bio/Molecular Science and Engineering, 4555 Overlook Avenue SW, Washington, DC 20375-5348, USA. Tel: +1 703 696 4504; Fax: +1 703 696 1212; Email: chrisel@onr.navy.mil
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