Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1995-02-22
1997-11-11
Jones, W. Gary
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 911, 435 9152, 435810, 536 243, 935 77, 935 78, C12P 1934, C12Q 168, C07H 2104
Patent
active
056862725
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to methods and kits for amplifying and detecting specific RNA sequences. In particular, the invention relates to methods for producing complementary DNA (cDNA) by reverse transcription of RNA, and amplification of the DNA sequences. The analysis of the amplified material facilitates the detection of pathogens and disease states associated with the presence of particular nucleic acid sequences, so the present invention is important in medical diagnostic procedures.
BACKGROUND
Nucleic acid amplification techniques are established as powerful tools for detecting small amounts of DNA or RNA which previously were undetectable by standard nucleic acid hybridization methods. DNA amplification most commonly employs the polymerase chain reaction (PCR) as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 or the ligase chain reaction (LCR) as described in EP-A-320 308 and EP-A-439 182. The entire disclosure of each of these publications is incorporated herein by reference.
When coupled with reverse transcription, PCR permits the amplification and detection of minute amounts of RNA as described in PCR Protocols: A Guide to Methods and Amplifications, Academic Press, Inc., (1990). The PCR process is discussed further in WO 91/0994, which describes a one-enzyme system that can amplify RNA. A thermostable DNA polymerase having reverse transcriptase activity is reported. The reverse transcriptase activity makes a cDNA copy of the RNA and the cDNA is amplified by PCR, using the same enzyme and reagents.
Efforts to avoid amplifying contaminating DNA are disclosed by Shuldiner et al., in published U.S. patent application Ser. No. 07/504,591 (NTIS) published May 14, 1991 for RNA template-specific PCR.
The present invention provides a method to amplify RNA using the LCR. It utilizes a combination of oligonucleotide probes and amplification methods which enhance the sensitivity and reliability of RNA amplification and detection with LCR.
SUMMARY OF THE INVENTION
The present invention relates to methods and kits useful for amplifying and detecting ribonucleic acid (RNA) from a multitude of sources. In a first aspect, the invention provides a method of amplifying a known RNA target sequence present in a biological sample, said method comprising: oligonucleotide probe which is hybridizable to a first segment of the known target RNA; the RNA target so that a cDNA segment is produced having at its 5' end said first probe and at its extended 3' end a nucleotide sequence complementary to a second segment of the target RNA, said reverse transcription being limited to the addition of not more than about 30 nucleotides; probe, said second probe having a 3' end hybridizable to the extended cDNA segment of the first probe, but substantially not hybridizable to said first probe when it is unextended; probe to the 3' terminus of said second probe, with the proviso that if said second or third probe is modified, it is corrected prior to ligation of the third probe to the second probe; and covalently attached to the 3' terminus of said first probe and complementary to said second probe; and elongated first probe complex.
Preferably, the length of the cDNA extension of the first probe is limited to a predetermined length by providing a pool of less than all four nucleoside triphosphate types. In this way, extension is terminated at the stopbase which calls for an omitted nucleotide.
An important part of this invention is the formation of a DNA copy from the RNA that is long enough to support amplification. The method of the invention provides several ways of accomplishing this, it being particularly important that the second probe hybridize with the first probe substantially only when the first probe has been extended on the RNA target. Ideally, the second and extended first probes hybridize together for only a relatively short portion at their respective 3' ends, leaving relatively large 5' overhangs. The 5' overhangs are then used to complete the formation of a full length DNA product. Th
REFERENCES:
patent: 4988617 (1991-01-01), Landegren et al.
patent: 5185243 (1993-02-01), Ullman et al.
patent: 5427930 (1995-06-01), Birkenmeyer et al.
Blumberg, B. M., et al., "Human Immunodeficiency Virus Type 1 nef Quasispecies in Pathological Tissue", Journal of Virology, 66(9):5256-5264 (1992).
Marshall, R. L., et al., "Detection of HCV RNA by the Asymmetric Gap Ligase Chain Reaction", 6189 PCR Methods and Applications, 2:80-84 (1994).
Innis et al., eds. "PCR Protocols: A Guide to Methods and Applications," (1990) Academic Press, Inc. pp. 21-27.
Sokolov Nuc. Acids Res. 18(12):3671, 1990.
Carrino John J.
Marshall Ronald L.
Sustachek Joann C.
Abbott Laboratories
Brainard Thomas D.
Jones W. Gary
Tran Paul B.
Yasger Paul D.
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