The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.

The effect of phosphorothioate bonds on the hydrolytic activity of the S' --~ 3 ' double-strand-specific T7 gene 6 exonuclease was studied. Double-stranded DNA substrates containing one phosphorothioate residue at the 5' end were found to be hydrolyzed by this enzyme as efficiently as unmodified ones. The enzyme activity was, however, completely inhibited by the presence of four phosphorothioates. On the basis of these results, a method for the conversion of double-stranded PCR products into full-length, singlestranded DNA fragments was developed. In this method, one of the PCR primers contains four phosphorothioates at its 5' end, and the opposite strand primer is unmodified. Following the amplification, the double-stranded product is treated with T7 gene 6 exonuclease. The phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed. When the phosphorothioated PCR primer is S' biotinylated, the singlestranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate. We also describe a simple and efficient method for the immobilization of relatively short oligonucleotides to microtiter plates with a hydrophllic surface in the presence of salt. P C R (1) is currently the most widely used DNA amplification method. It serves as the first step in many genetic analysis methods. PCR normally produces double-stranded products. For a number of applications the doublestranded PCR product must be converted to the single-stranded form. Such applications include the sequencing of PCR products or their use as hybridization probes. In this paper we show that if one of the 5' ends of a double-stranded PCR product is selectively protected from the hydrolytic action of T7 gene 6 exonuclease, the opposite strand can be hydrolyzed completely by this enzyme. Singlestranded PCR product is thereby generated with high efficiency. We have applied this approach to the development of a new, nonelectrophoretic, ELISA-type detection method for PCR products. There are a number of analytical methods available for the detection of PCR products. The most specific of these methods, and at the same time one of the most labor-intensive, requires the polyacrylamide or agarose gel electrophoresis of the PCR product, followed by blotting onto a membrane and hybridization to a detectably labeled PCR product-specific probe. For more routine applications, the blotting and hybridization steps are omitted, but the need for gel electrophoresis still presents a bottleneck when a large number of PCR products have to be analyzed. There is a need for more efficient, rapid, nonelectrophoretic, nonradioactive methods for the detection of PCR products. Currently, 96-well polystyrene plates are widely used in solid-phase immunoassays, and several PCR product detection methods that use plates as a solid support have been described. (~-12) The most specific of these methods require the immobilization of a suitable oligonucleotide probe into the microtiter wells followed by the capture of the PCR product by hybridization and colorimetric detection of a suitable hapten. The method described in this paper also employs the widely used 96-well plate format. Thus, it is very well suited for automation and large-scale application.