Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format

Kwoh, D. Y., Davis, G. R., Whitfield, K. M., Chappelle, H. L., DiMichelle, L. J., Gingeras, T. R. (1989) Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proceedings of the National Academy of Sciences of the United States of America, 86 (4). pp. 1173-1177. ISSN 00278424 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/2919166

Abstract

The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 106-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 106 uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.

Item Type: Paper
Additional Information:
Uncontrolled Keywords: nucleic acid rna polymerase gene amplification genetic engineering human human cell human immunodeficiency virus 1 priority journal Cell Line Cell Transformation, Viral Genes, Viral HIV-1 Nucleic Acid Hybridization Oligonucleotide Probes RNA Viral Transcription Genetic
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > transcription
Investigative techniques and equipment
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA replication
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene amplification
organism description > virus
CSHL Authors:
Communities: CSHL labs > Gingeras lab
Depositing User: CSHL Librarian
Date: 1989
Date Deposited: 14 Mar 2012 14:23
Last Modified: 31 Mar 2014 13:42
PMCID: PMC286648
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25243

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