Post-PCR sterilization: development and application to an HIV-1 diagnostic assay.

We have developed an effective post-PCR sterilization process and have applied the procedure to a diagnostic assay for HIV-1. The method, which is based on isopsoralen photochemistry, satisfies both the inactivation and hybridization requirements of a practical sterilization process. The key feature of the technique is the use of isopsoralen compounds which form covalent photochemical adducts with DNA. These covalent adducts prevent subsequent extension of previously amplified sequences (amplicons) by Taq polymerase. Isopsoralens have minimal inhibitory effect on the PCR, are activated by long wavelength ultraviolet light, provide sufficient numbers of covalent adducts to impart effective sterilization, modify the amplified sequence such that it remains single-stranded, and have little effect on subsequent hybridization. The sterilization procedure can be applied to a closed system and is suitable for use with commonly used detection formats. The photochemical sterilization protocol we have devised is an effective and pragmatic method for eliminating the amplicon carryover problem associated with the PCR. While the work described here is limited to HIV-1, proper use of the technique will relieve the concern associated with carryover for a wide variety of amplicons, especially in the clinical setting.

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.