Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction.
We describe a photochemical procedure for the sterilization of polynucleotides that are created by the Polymerase Chain Reaction (PCR). The procedure is based upon the blockage of Taq DNA polymerase when it encounters a photochemically modified base in a polynucleotide strand. We have discovered reagents that can be added to a PCR reaction mixture prior to amplification and tolerate the thermal cycles of PCR, are photoactivated after amplification, and damage a PCR strand in a manner that, should the damaged strand be carried over into a new reaction vessel, prevent it from functioning as a template for the PCR. These reagents, which are isopsoralen derivatives that form cyclobutane adducts with pyrimidine bases, are shown to stop Taq polymerase under conditions appropriate for the PCR process. We show that effective sterilization of PCR products requires the use of these reagents at concentrations that are tailored to the length and sequence of the PCR product and the level of amplification of the PCR protocol.
Cimino
G D
GD
HRI Research, Inc., Berkeley, CA 94710.
Metchette
K C
KC
Tessman
J W
JW
Hearst
J E
JE
Isaacs
S T
ST
D001483:Base Sequence; D004247:DNA; D011564:Furocoumarins / Q000737:chemistry; D005784:Gene Amplification; D007700:Kinetics; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D009841:Oligonucleotides; D010777:Photochemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase
D011564:Furocoumarins; D019384:Nucleic Acid Synthesis Inhibitors; D009841:Oligonucleotides; D004247:DNA; C011659:angelicin; D019914:Taq Polymerase