PCR HOME:
PCR HISTORY:
FOUNDATIONAL PAPERS
PCR PROJECT:
A. Basic Research (Seminal reports of PCR Research/Discovery)
Saiki R, K.; Scharf S; Faloona F; Mullis K. B; Horn G. T; Erlich H. A.; Arnheim N., Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science, 1985 Dec 20, 230(4732):1350-4.
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 thanMullis K. B; Faloona F. A; Scharf S; Saiki R. K; Horn G; Erlich H. A., Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology, 1986
Scharf S. J; Horn G. T; Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science, 1986 Sep 5, 233(4768):1076-8.
Abstract: A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human beta-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ alpha locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5' ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.
Saiki R. K; Bugawan T. L; Horn G. T; Mullis K. B; Erlich H. A. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature, 1986 Nov 13-19, 324(6093):163-6.
Abstract: Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.
Mullis K. B; Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology, 1987, 155:335-50.
Saiki R. K; Gelfand D. H; Stoffel S; Scharf S. J; Higuchi R; Horn G. T; Mullis K. B; Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 1988 Jan 29, 239(4839):487-91.
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.
Lawyer F. C; Stoffel S; Saiki R. K; Myambo K; Drummond R; Gelfand D. H. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. Journal of Biological Chemistry, 1989 Apr 15, 264(11):6427-37.
Abstract: The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction method for amplifying DNA. We report the cloning and expression of Taq DNA polymerase in Escherichia coli. From a lambda gt11:Taq library we identified a Taq DNA fragment encoding an epitope of Taq DNA polymerase via antibody probing. The fusion protein from the lambda gt11:Taq candidate selected an antibody from an anti-Taq polymerase polyclonal antiserum which reacted with Taq polymerase on Western blots. We used the lambda gt11 clone to identify Taq polymerase clones from a lambda Ch35:Taq library. The complete Taq DNA polymerase gene has 2499 base pairs. From the predicted 832-amino acid sequence of the Taq DNA polymerase gene, Taq DNA polymerase has significant similarity to E. coli DNA polymerase I. We subcloned and expressed appropriate portions of the insert from a lambda Ch35 library candidate to yield thermostable, active, truncated, or full-length forms of the protein in E. coli under control of the lac promoter.
Guyer R. L; Koshland D. E ,Jr. The Molecule of the Year. Science, 1989 Dec 22, 246(4937):1543-6.
PCR Bibliography, Perkin Elmer Cetus
B. Reviews of PCR Research and PCR applications
Appenzeller T. Democratizing the DNA sequence., Science, 1990 Mar 2, 247(4946).
Erlich, H. A; Gelfand, D; Sninsky, J. J. Recent Advances in the Polymerase Chain Reaction., Science, 1991, v.252, n.5013, 1643-1651.
Arnheim, N. Polymerase Chain Reaction Strategy. ANNUAL REVIEW OF BIOCHEMISTRY, VOL. 61. XIV+1359P. , 1992. p. 131-156.
White T. J. The future of PCR technology: diversification of technologies and applications. Trends Biotechnology ,1996 Dec, 14(12):478-83.
Abstract: The polymerase chain reaction has had a major impact o research in molecular biology, on the human genome project and on the diagnosis of disease. In the future, this powerful technique will be increasingly applied to the study of individual genomic variation and to assess the genetic consequences of ecological and demographic events on past and contemporary populations of many organisms.
II. Applications of PCR "Basics"
Olson M; Hood L; Cantor C; Botstein D. A common language for physical mapping of the human genome Science, 1989 Sep 29, 245(4925):1434-5.
Paabo, S. Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic amplification. Proceedings of the National Academy of Sciences of the United States of America, 1989, v.86, n.6.
Abstract: Several chemical and enzymatic properties were examined in the DNA extracted from dry remains of soft tissues that vary in age from 4 to 13,000 years and represent four species, including two extinct animals (the marsupial wolf and giant ground sloth). The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, which primarily manifested themselves as modifications of pyrimidines and sugar residues as well as baseless sites and intermolecular cross-links. This renders molecular cloning difficult. However, the polymerase chain reaction can be used to amplify and study short mitochondrial DNA sequences that are of anthropological and evolutionary significance. This opens up the prospect of performing diachronical studies of molecular evolutionary genetics.
Paabo, S; Higuchi, R. G; Wilson, A. C. Ancient Dna and the Polymerase Chain Reaction the Emerging Field of Molecular Archaeology. Journal of Biological Chemistry , 1989
Kocher T. D; Thomas W. K; Meyer A; Edwards S. V; Paabo S; Villablanca F. X; Wilson A. C. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the United States of America , 1989 Aug, 86(16):6196-200.
Abstract: With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments of mtDNA from more than 100 animal species, including mammals, birds, amphibians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nanogram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility of these primers offers opportunities for phylogenetic and population research.
Paabo, S; Higuchi, R. G; Wilson, A. C. Ancient Dna and the Polymerase Chain Reaction the Emerging Field of Molecular Archaeology. Journal of Biological Chemistry, 1989
Saiki, R. K; Walsh, P. S; Levenson, C. H; Erlich, H. A. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proceedings of the National Academy of Sciences of the United States of America, 1989: 6230-6234.
Abstract: The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean.beta.-thalassemia mutations (nine alleles).
White T. J; Madej R; Persing D. H. The polymerase chain reaction: clinical applications. Advances in Clinical Chemistry, 1992, 29:161-96.
Leeflang, E. P; Zhang, L; Tavare, S; Hubert, R; Srinidhl, J; MacDonald, M. E; Myers, R H; De Young, M; Wexler, N S; Gusella, J F; and others. Single sperm analysis of the trinucleotide repeats in the Huntington's disease gene: Quantification of the mutation frequency spectrum. Human Molecular Genetics, 1995, v.4, n.9. 1519-1526.
Abstract: The CAG triplet repeat region of the Huntington's disease gene was amplified in 923 single sperm from three affected and two normal individuals. Averagesize alleles (1518 repeats) showed only three contraction mutations among 475 sperm (0.6%). A 30 repeat normal allele showed an 11% mutation frequency. The mutation frequency of a 36 repeat intermediate allele was 53% with 8% of all gametes having expansions which brought the allele size into the HD disease range ( greater than or equal to 38 repeats). Disease alleles (3851 repeats) showed a very high mutation frequency (9299%). As repeat number increased there was a marked elevation in the frequency of expansions, in the mean number of repeats added per expansion and the size of the largest observed expansion. Contraction frequencies also appeared to increase with allele size but decreased as repeat number exceeded 36. Our sperm typing data are of a discrete nature rather than consisting of smears of PCR product from pooled sperm. This allowed the observed mutation frequency spectra to be compared to the distribution calculated using discrete stochastic models based on current molecular ideas of the expansion process. An excellent fit was found when the model specified that a random number of repeats are added during the progression of the polymerase through the repeated region.
III. Technological Variations of PCR "Basics"
Holland, P. M; Abramson, R. D; Watson, R; Gelfand, D. H. Detection of specific polymerase chain reaction product by utilizing the 5'.fwdarw.3' exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America, 1991, v.88, n.16.
Abstract: The 5'-.forward arrow. 3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'.forward arrow. 3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.
Higuchi R; Dollinger G; Walsh P. S; Griffith R. Simultaneous amplification and detection of specific DNA sequences., Biotechnology, 1992 Apr, 10(4).
Abstract: We have enhanced the polymerase chain reaction (PCR) such that specific DNA sequences can be detected without opening the reaction tube. This enhancement requires the addition of ethidium bromide (EtBr) to a PCR. Since the fluorescence of EtBr increases in the presence of double-stranded (ds) DNA an increase in fluorescence in such a PCR indicates a positive amplification, which can be easily monitored externally. In fact, amplification can be continuously monitored in order to follow its progress. The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification both simplifies and improves PCR and may facilitate its automation and more widespread use in the clinic or in other situations requiring high sample throughput.
Erlich, H.A.; Gelfand, D. H.; Saiki, R. K. Specific DNA Amplification, Nature, 1988, Feburary 4, V. 331, 461-462
Bugawan, T. L; Saiki, R. K; Levenson, C. H; Watson, R. W; Erlich, H. A. The Use of Non-Radioactive Oligonucleotide Probes To Analyze Enzymatically Amplified Dna for Prenatal Diagnosis and Forensic Hla Typing., Bio-Technology, 1988
Kinzler . W; Vogelstein B. Whole genome PCR: application to the identification of sequences bound by gene regulatory proteins., Nucleic Acids Research, 1989 May 25, 17(10):3645-53.
Abstract: A strategy is described that allows the isolation of DNA sequences that can bind to gene regulatory proteins. Total genomic DNA is first converted to a form that is suitable for amplification by the polymerase chain reaction (Whole Genome PCR), and the DNA sequences of interest are selected by binding to the regulatory protein and immune precipitation. Because sequences recovered from the selection step can be amplified by PCR, the selection process can be designed for maximum enrichment with little concern about recovery. Furthermore, the selection process can be repeated as often as necessary. Sequences recovered after amplification can be cloned and/or used as hybridization probes. As a test of this strategy, we selected human sequences that bound to Xenopus transcription factor IIIA (TFIIIA). Seven clones were isolated that were on the average 94% identical to the previously described 61 bp binding site of TFIIIA. This strategy could be adapted to isolate sequences that can be selected by any physical or biological method.
C. Amplifying unknown sequences
Erlich, H.A.; Gelfand, D. H.; Saiki, R. K. Specific DNA Amplification, Nature, 1988, Feburary 4, V. 331, 461-462
Bugawan, T. L; Saiki, R. K; Levenson, C. H; Watson, R. W; Erlich, H. A. The Use of Non-Radioactive Oligonucleotide Probes To Analyze Enzymatically Amplified Dna for Prenatal Diagnosis and Forensic Hla Typing. Bio-Technology, 1988
Kinzler . W; Vogelstein B. Whole genome PCR: application to the identification of sequences bound by gene regulatory proteins. Nucleic Acids Research, 1989 May 25, 17(10):3645-53.
Abstract: A strategy is described that allows the isolation of DNA sequences that can bind to gene regulatory proteins. Total genomic DNA is first converted to a form that is suitable for amplification by the polymerase chain reaction (Whole Genome PCR), and the DNA sequences of interest are selected by binding to the regulatory protein and immune precipitation. Because sequences recovered from the selection step can be amplified by PCR, the selection process can be designed for maximum enrichment with little concern about recovery. Furthermore, the selection process can be repeated as often as necessary. Sequences recovered after amplification can be cloned and/or used as hybridization probes. As a test of this strategy, we selected human sequences that bound to Xenopus transcription factor IIIA (TFIIIA). Seven clones were isolated that were on the average 94% identical to the previously described 61 bp binding site of TFIIIA. This strategy could be adapted to isolate sequences that can be selected by any physical or biological method.
Scharf S. J; Horn G. T; Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science, 1986 Sep 5, 233(4768):1076-8.
Abstract: A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human beta-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ alpha locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5' ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.
Saiki R. K; Bugawan T. L; Horn G. T; Mullis K. B; Erlich H. A. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature, 1986 Nov 13-19, 324(6093):163-6.
Abstract: Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.
Saiki R. K; Gelfand D. H; Stoffel S; Scharf S. J; Higuchi R; Horn G. T; Mullis K. B; Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 1988 Jan 29, 239(4839):487-91.
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.
Erlich, H.A.; Gelfand, D. H.; Saiki, R. K. Specific DNA Amplification Nature, 1988, Feburary 4, V. 331, 461-462
White T. J; Arnheim N; Erlich H. A. The polymerase chain reaction. Trends in Genetics, 1989 Jun, 5(6):185-9.
Abstract: The polymerase chain reaction (PCR) is a powerful new method for 'in vitro cloning'. It can selectively amplify a single molecule of template DNA several millionfold in a few hours and has made possible new approaches to problems in molecular genetics, evolutionary biology, and development.
Arnheim N; Li H. H; Cui X. F. PCR analysis of DNA sequences in single cells: single sperm gene mapping and genetic disease diagnosis. Genomics, 1990 Nov, 8(3).
Arnheim, N; White, T. J; Rainey, W. E. Application of PCR: Organismal and Population Biology Polymerase Chain Reaction Can Produce Large Quantities of Specific Dna from Small Degraded and Impure Samples. Bioscience, 1990, v.40, n.3. 174-182.
Kellogg, D. E; Sninsky, J. J; Kwok, S. Quantitation of HIV-1 proviral DNA relative to cellular DNA by the polymerase chain reaction. Analytical Biochemistry, 1990, v.189, n.2. 202-208.
Abstract: We developed a quantitative assay for human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences using the polymerase chain reaction (PCR). The relative copy numbers of HIV-1 proviral DNA molecules were determined by coamplification of an HIV-1 gag sequence and a portion of the DQ.alpha. locus of the histocompatibility (HLA) region. Because of the disparity in the copy number of cellular and HIV-1 templates, an attenuation in the efficiency of the HLA amplification was required to achieve simultaneous amplification and quantification of both target sequences. The HIV-1 and HLA amplified products were detected by hybridization with radioactively labeled probes and the amount of probe bound to each product was determined with a radioanalytic system. Standard curves were generated by plotting the HIV-1 and HLA signals made against known copies of each target present prior to amplification. The copies of HIV-1 target relative to the number of cells in a given sample were determined by interpolation from standard curves. The procedure described here is generally applicable to the quantitation of other retroviruses.
Arnheim, N. Polymerase Chain Reaction Strategy. ANNUAL REVIEW OF BIOCHEMISTRY, VOL. 61. XIV+1359P., 1992. p. 131-156.
White T. J; Madej R; Persing D. H. The polymerase chain reaction: clinical applications. Advances in Clinical Chemistry, 1992, 29:161-96.
Higuchi R; Fockler C; Dollinger G; Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology N Y, 1993 Sep, 11(9):1026-30.
Abstract: We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.