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

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Isaacs ST,Tessman JW,Metchette KC,Hearst JE,Cimino GD

doi

10.1093/nar/19.1.109

subject

Has Abstract

pub_date

1991-01-11 00:00:00

pages

109-16

issue

1

eissn

0305-1048

issn

1362-4962

journal_volume

19

pub_type

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