Showing papers on "Multiplex polymerase chain reaction published in 1986"

Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction

Abstract: The discovery of specific restriction endonucleases (Smith and Wilcox 1970) made possible the isolation of discrete molecular fragments of naturally occurring DNA for the first time. This capability was crucial to the development of molecular cloning (Cohen et al. 1973); and the combination of molecular cloning and endonuclease restriction allowed the synthesis and isolation of any naturally occurring DNA sequence that could be cloned into a useful vector and, on the basis of flanking restriction sites, excised from it. The availability of a large variety of restriction enzymes (Roberts 1985) has significantly extended the utility of these methods. The de novo organic synthesis of oligonucleotides and the development of methods for their assembly into long double-stranded DNA molecules (Davies and Gassen 1983) have removed, at least theoretically, the minor limitations imposed by the availability of natural sequences with fortuitously unique flanking restriction sites. However, de novo synthesis, even with automated equipment, is not easy; it is often fraught with peril due to the inevitable indelicacy of chemical reagents (Urdea et al. 1985; Watt et al. 1985; Mullenbach et al. 1986), and it is not capable of producing, intentionally, a sequence that is not yet fully known. We have been exploring an alternative method for the synthesis of specific DNA sequences (Fig. 1). It involves the reciprocal interaction of two oligonucleotides and the DNA polymerase extension products whose synthesis they prime, when they are hybridized to different strands of a DNA template in a relative orientation such that their extension products overlap. The method consists of repetitive cycles of denaturation, hybridization, and polymerase extension and seems not a little boring until the realization occurs that this procedure is catalyzing a doubling with each cycle in the amount of the fragment defined by the positions of the 5' ends of the two primers on the template DNA, that this fragment is therefore increasing in concentration exponentially, and that the process can be continued for many cycles and is inherently very specific. The original template DNA molecule could have been a relatively small amount of the sequence to be synthesized (in a pure form and as a discrete molecule) or it could have been the same sequence embedded in a much larger molecule in a complex mixture as in the case of a fragment of a single-copy gene in whole human DNA. It could also have been a single-stranded DNA molecule or, with a minor modification in the technique, it could have been an RNA molecule. In any case, the product of the reaction will be a discrete double-stranded DNA molecule with termini corresponding to the 5' ends of the oligonucleotides employed. We have called this process polymerase chain reaction or (inevitably) PCR. Several embodiments have been devised that enable one not only to extract a specific sequence from a complex template and amplify it, but also to increase the inherent specificity of this process by using nested primer sets, or to append sequence information to one or both ends of the sequence as it is being amplified, or to construct a sequence entirely from synthetic fragments.