• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Aug 15, 1991; 88(16): 7276–7280.
PMCID: PMC52277

Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

Abstract

The 5'----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'----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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, 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):1350–1354. [PubMed]
  • Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Innis MA, Myambo KB, Gelfand DH, Brow MA. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. [PMC free article] [PubMed]
  • Cozzarelli NR, Kelly RB, Kornberg A. Enzymic synthesis of DNA. 33. Hydrolysis of a 5'-triphosphate-terminated polynucleotide in the active center of DNA polymerase. J Mol Biol. 1969 Nov 14;45(3):513–531. [PubMed]
  • Lundquist RC, Olivera BM. Transient generation of displaced single-stranded DNA during nick translation. Cell. 1982 Nov;31(1):53–60. [PubMed]
  • Miller H. Practical aspects of preparing phage and plasmid DNA: growth, maintenance, and storage of bacteria and bacteriophage. Methods Enzymol. 1987;152:145–170. [PubMed]
  • Hart C, Chang SY, Kwok S, Sninsky J, Ou CY, Schochetman G. A replication-deficient HIV-1 DNA used for quantitation of the polymerase chain reaction (PCR). Nucleic Acids Res. 1990 Jul 11;18(13):4029–4030. [PMC free article] [PubMed]
  • Jay E, Bambara R, Padmanabhan R, Wu R. DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping. Nucleic Acids Res. 1974 Mar;1(3):331–353. [PMC free article] [PubMed]
  • Kwok S, Ehrlich G, Poiesz B, Kalish R, Sninsky JJ. Enzymatic amplification of HTLV-I viral sequences from peripheral blood mononuclear cells and infected tissues. Blood. 1988 Oct;72(4):1117–1123. [PubMed]
  • Rayfield M, De Cock K, Heyward W, Goldstein L, Krebs J, Kwok S, Lee S, McCormick J, Moreau JM, Odehouri K, et al. Mixed human immunodeficiency virus (HIV) infection in an individual: demonstration of both HIV type 1 and type 2 proviral sequences by using polymerase chain reaction. J Infect Dis. 1988 Dec;158(6):1170–1176. [PubMed]
  • Ott J, Eckstein F. Protection of oligonucleotide primers against degradation by DNA polymerase I. Biochemistry. 1987 Dec 15;26(25):8237–8241. [PubMed]
  • Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 1990 Feb 25;18(4):999–1005. [PMC free article] [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Spector SA, Rua JA, Spector DH, McMillan R. Detection of human cytomegalovirus in clinical specimens by DNA-DNA hybridization. J Infect Dis. 1984 Jul;150(1):121–126. [PubMed]
  • Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6230–6234. [PMC free article] [PubMed]
  • Chehab FF, Kan YW. Detection of specific DNA sequences by fluorescence amplification: a color complementation assay. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9178–9182. [PMC free article] [PubMed]
  • Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 1988 Dec 9;16(23):11141–11156. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...