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.

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Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.

AFLP: a new technique for DNA fingerprinting.

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.

ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts.

Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.

https://science.sciencemag.org/content/sci/230/4732/1350.full.pdf

Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.

The present invention is directed to a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, and extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired.

Process for amplifying nucleic acid sequences

Publisher Summary This chapter discusses the specific synthesis of deoxyribonucleic acid (DNA) in vitro through the medium of a polymerase-catalyzed chain reaction. A method whereby a nucleic acid sequence can be exponentially amplified in vitro is described in the chapter. The same method can be used to alter the amplified sequence or to append new sequence information to it. It is necessary that the ends of the sequence be known in sufficient detail that oligonucleotides can be synthesized, which will hybridize to them and that a small amount of the sequence be available to initiate the reaction. The oligonucleotides are complementary to different strands of the desired sequence and at relative positions along the sequence such that the DNA polymerase extension product of the one, when denatured, can serve as a template for the other and vice versa. Oligonucleotides were synthesized using an automated DNA synthesis machine (Biosearch, Inc., San Rafael, California) using phosphoramidite chemistry. “Mispriming"” can be usefully employed to make intentional in vitro mutations or to add sequence information to one or both ends of a given sequence. The chapter explores the possibility of utilizing a heat-stable DNA polymerase so as to avoid the need for addition of new enzyme after each cycle of thermal denaturation

Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.

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.

Kary B. Mullis

Papers

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.

Process for amplifying nucleic acid sequences

Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.

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