title: Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions.
abstract: 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.
authors.affiliation: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington, DC 20375-5348, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Andreadis
authors.forename: L A
authors.initials: LA
authors.lastname: Chrisey
mesh_terms: D000818:Animals; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014981:Xenopus
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; C064517:gp23 protein, Bacteriophage T4; D049452:Green Fluorescent Proteins