Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.
TL;DR: A method whereby a nucleic acid sequence can be exponentially amplified in vitro is described in the chapter, and the possibility of utilizing a heat-stable DNA polymerase is explored so as to avoid the need for addition of new enzyme after each cycle of thermal denaturation.
Abstract: 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
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TL;DR: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Abstract: 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.
17,663 citations
TL;DR: The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity that allows the specific co-amplification of high numbers of restriction fragments.
Abstract: 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.
12,960 citations
TL;DR: In this paper, two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit were proposed, which were intended to be specific to fungi and basidiomycetes, respectively.
Abstract: 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.
8,128 citations
TL;DR: In this paper, complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends, and these fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the three' extension of the complementary strand.
7,528 citations
TL;DR: The generality of the arbitrarily primed PCR method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa.
Abstract: Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).
5,472 citations
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TL;DR: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia, using primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase of target DNA copies.
Abstract: 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.
9,107 citations