Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication

Guatelli, J. C., Whitfield, K. M., Kwoh, D. Y., Barringer, K. J., Richman, D. D., Gingeras, T. R. (1990) Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proceedings of the National Academy of Sciences of the United States of America, 87 (5). pp. 1874-1878. ISSN 00278424 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/2217213

Abstract

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.

Item Type: Paper
Uncontrolled Keywords: reserve transcriptase RNase H T7 RNA polymerase rna directed dna polymerase article gene amplification genetic engineering nucleic acid amplification priority journal retrovirus rna amplification Base Sequence Cell Line Cloning Molecular DNA, Viral HIV-1 Human Kinetics Models Biological Molecular Sequence Data Nucleic Acid Amplification Techniques Nucleic Acid Hybridization Oligonucleotide Probes RNA Viral Transcription, Genetic Virus Replication unidentified retrovirus
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA replication
organism description > virus
CSHL Authors:
Communities: CSHL labs > Gingeras lab
Depositing User: CSHL Librarian
Date: 1990
Date Deposited: 13 Mar 2012 16:56
Last Modified: 28 Mar 2014 20:55
PMCID: PMC516580
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25250

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