Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-06-18
2001-05-22
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S195000, C536S024300
Reexamination Certificate
active
06235472
ABSTRACT:
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:
(i) The reaction is inherently very specific. Ligation reactions are based 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.
(ii) The ligation reaction can be employed to assess the accuracy of 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.
(iii) The act of ligation creates a unique molecule, not previously present 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 which has a repeated sequence complementary to the probe and at least partially to the template. The amplification product is then detected either via a labelled nucleic acid triphosphate incorporated in the amplification, or by an added labelled nucleic acid probe capable of hybridizing to the amplification product.
While this prior art technique comprises a ligase-based detection step, which as discussed above, per se may provide for high specificity, the specificity of the method will rather depend on the specificity in the generation and detection of the amplified product.
SUMMARY OF THE INVENTION
By contrast, the present invention provides a method based upon probe circularization but in which the potential specificity and binding stability of a circularizable DNA probe is fully utilized.
Thus, in accordance with the present inventive concept, efficient use is made of the fact that a probe, designed to be circularized in the presence of a target sequence, may be caused to close around the target-containing nucleic acid strand or strands, for example DNA or RNA, such that the cyclic probe will interlock with and thereby be efficiently linked to the target nucleic acid to be detected. In other words, because of the helical nature of double-stranded nucleic acids, such as DNA, circularized probes will be wound around the target strand, topologically connecting probes to target molecules through catenation, in a manner similar to “padlocks”. Such covalent catenation of probe molecules to target sequences results in the formation of a hybrid that resists extreme washing conditions, serving to reduce non-specific signals in genetic assays, as will be further described below. Any probes hybridizing in a non-specific manner may therefore be efficiently removed by subjecting the target to non-hybridizing conditions and/or exonuclease activity. Provided that the circular probe is properly detectable, such as suitably labelled, it may then be detected to indicate the presence of the target molecule. Although the target is not amplified, highly sensitive detection is possible through efficient reduction of non-specific signals.
In a variant of the above strategy, the probe is designed to hybridize to the target sequence with an interspace between the probe ends, and at least one other, or intermediate, probe is provided which is designed to hybridize with the interspatial target sequence. The probes are then interconnected to form a cyclized molecule.
In another variant, the probe is designed to hybridize to the target sequence with a small gap of from one to a few nucleotides between the probe ends. This gap is then filled by an enzyme-assisted extension reaction, such as by DNA polymerase, prior to connecting the probe ends with each other as is per se known in the art. It is readily appreciated that increased specificity may be obtained if such a gap does not contain all four nucleotides.
Both of the above variants will provide for increased specificity of the detection.
This proposed method of the invention, while having a higher specificity, is simpler to perform than the prior art method discussed above. Further, the novel method may be performed with even very short synthetic probes since only part of the probe molecule needs to form a rigid double-stranded DNA molecule with the target molecule, whereas the rest of the probe molecule may be highly flexible, optionally branched single-stranded DNA or any other spacer material. By contrast, a probe that will be replicated, as in the prior art technique, would be expected to require a length in excess of 100 nucleotides and with no non-nucleotide segment and no branced positions.
One aspect of the present invention therefore relates to an assay method of detecting a target nucleic acid sequence in a sample by contacting the sample with a detectable probe to hybridize the probe to the target sequence,
Kwiatkowski Marek
Landegren Ulf
Arthur Lisa B.
Ronning, Jr. Royal N.
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