A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

The 5' -* 3' exonuclease activity of the ther-

No abstract

The polymerase chain reaction (PCR) has dramatically altered how molecular studies are conducted as well as what questions can be asked. In addition to simplifying molecular tasks typically carried out with the use of recombinant DNA technology, PCR has allowed a spectrum of advances ranging from the identification of novel genes and pathogens to the quantitation of characterized nucleotide sequences. PCR can provide insights into the intricacies of single cells as well as the evolution of species. Some recent developments in instrumentation, methodology, and applications of the PCR are presented in this review.

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products >1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2'-deoxyguanosine was used to sequence through G+C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.DNA sequencing by the Sanger dideoxynucleotide method (1) has undergone significant refinement in recent years, including the development of additional vectors (2), base analogs (3, 4), enzymes (5), and instruments for partial automation of DNA sequence analysis (6-8). The basic procedure involves (i) hybridizing an oligonucleotide primer to a suitable single-or denatured double-stranded DNA template; (ii) extending the primer with DNA polymerase in four separate reaction mixtures, each containing one alabeled dNTP, a mixture of unlabeled dNTPs, and one chain-terminating ddNTP; (iii) resolving the four sets of reaction products on a high-resolution polyacrylamide/urea gel; and (iv) producing an autoradiographic image of the gel, which can be examined to infer the DNA sequence. The current commercial instruments address nonisotopic detection and computerized data collection and analysis. The ultimate success of large-scale sequencing projects will depend on further improvements in the speed and automation of the technology. These include automating the preparation of DNA templates and performing the sequencing reactions.One technique that appears to be ideally suited for automating DNA template preparation is the selective amplification of DNA by the polymerase chain reaction (PCR) (9). With this method, segments of single-copy genomic DNA can be amplified >10 million-fold with very high specificity and fidelity. The PCR product can then either be subcloned into a vector suitable for sequence analysis or, alternatively, purified PCR products can be sequenced (10)(11)(12)(13) Tween 20/0.5% Nonidet P-40), and 4 , p of H20. The labeling reaction mixture was incubated for 1 min at 37°C (see Fig. 3). Note: for sequencing with 5'-labeled primers, the addition of Abbreviations: c7GTP, 7-deaza-2'-deoxyguanosine 5'-triphosphate; PCR, polymerase chain reaction. *To whom reprint requests should be addressed. 9436The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.