Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-08-23
1999-02-16
Arthur, Lisa B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
935 77, 935 78, C12Q 168
Patent
active
058719210
DESCRIPTION:
BRIEF SUMMARY
This application was filed under 35 U.S.C. 371 from PCT/SE95/00163 on Feb. 16, 1995.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel method of detecting specific nucleic acid sequences as well as a detecting reagent and kit therefor.
2. Related Art
The isolation of enzymes that can join separate DNA molecules was fundamental to the development of recombinant DNA technology. Using these enzymes, called ligases, genetic elements derived from different genomes can be brought to be expressed together in an organism. Ligase-assisted reactions have also come to play an increasing role in gene detection. The principle employed in such reactions is that two probe-segments, complementary to target sequences situated in juxtaposition, are joined to a contiguous probe sequence through the agency of a DNA ligase. This is in contrast to more conventional detection schemes where the hybridization of single probes to target sequences is detected after removal of free probe molecules.
One type of ligase-assisted detection method is disclosed in U.S. Pat. No. 4,988,617, which relates to an assay for determining the nucleic acid sequence in a region of a nucleic acid test substance which has a known possible mutation in at least one target nucleotide position of the sequence. The assay comprises annealing two oligonucleotide probes to immediately adjacent segments of a substantially complementary test DNA or RNA molecule which contains the possible mutation(s) near the segment joint, and adding a linking agent, usually a ligase. The conditions are selected such that when the target nucleotide is correctly base paired, the probes are covalently joined, and if not correctly base paired due to a mismatching nucleotide(s) near the the segment joint, the probes are incapable of being covalently joined by the ligase. The presence or absence of linking is detected as an indication of the sequence of the target nucleotide.
A similar ligase-assisted detection method is disclosed in EP-A-185 494. In this method, however, the formation of a ligation product depends on the capability of two adjacent probes to hybridize under high stringency conditions rather than on the requirement of correct base-pairing in the joint region for the ligase to function properly as in the above U.S. Pat. No. 4,988,617. Other references relating to ligase-assisted detection are, e.g., EP-A-330 308, EP-A-324 616, EP-A-473 155, EP-A-336 731, U.S. Pat. No. 4,883,750 and U.S. Pat. No. 5,242,794.
The principal advantages of ligase-based detection strategies over other molecular genetic detection reactions are at least three-fold: on the coincidence of two separate probe sequences on a target sequence, and this is unlikely to occur in the absence of the appropriate target molecule even under non-stringent reaction conditions. For this reason the reaction is suitable in standardized, automated schemes. hybridization at positions immediately surrounding the junction between two probes. Due to the substrate requirements of ligases, terminally mismatched probes are ligated at a substantially reduced rate. In this manner, allelic sequence variants can be distinguished. in the-assay. This circumstance can be employed, for example, by taking advantage of the increased hybridization stability upon ligation.
JP-A-4262799 and JP-A-4304900 both disclose the use of ligation reactions combined with amplification reactions for detecting a target nucleic acid sequence in a specimen sample. The methodology comprises contacting the sample in the presence of a ligase with a straight chain probe polynucleotide, which has a sequence designed to be cyclized, or circularized, as the result of the presence of a target nucleic acid sequence. The cyclized polynucleotide is then used as a template in an enzymatic polymerization reaction. By adding a primer which is at least partially complementary to the cyclized probe together with a nucleic acid polymerase and nucleotide triphosphates, a single stranded chain nucleic acid is formed whi
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Kwiatkowski Marek
Landegren Ulf
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