Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-15
2001-08-07
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007100, C435S007200, C435S091200, C536S024300
Reexamination Certificate
active
06270965
ABSTRACT:
The present invention concerns a method and a device which enables an integrated amplification and detection of nucleic acids.
Heterogeneous methods for the detection of nucleic acids include the step of immobilizing the nucleic acid to be detected. This can for example take place directly by covalent binding of the nucleic acid to a solid phase or by coupling the nucleic acid to a partner of an affinity pair and binding the other partner of the affinity pair to a solid phase. A further method of coupling is also to immobilize the nucleic acid to be detected by means of hybridization to a solid-phase-bound so-called capture probe which is at least partially complementary to the nucleic acid to be detected. This capture probe can be bound or become bound to a solid phase in any desired manner.
Examples of methods in which nucleic acids to be detected are immobilized by means of capture probes are described in EP-A-0 078 139 for direct binding and in EP-A-0 192 168 for binding via an affinity pair. In the procedures described in these references the nucleic acid to be detected is hybridized with a detection probe which can also hybridize with the nucleic acid to be detected and, after removing excess detection probe, the hybrid formed is detected on the solid phase by means of a marker group of the detection probe. In order to detect nucleic acids which occur rarely in a sample, a target-specific amplification is usually carried out. Such a method in which for example a digoxigenin marker group is incorporated into the amplified nucleic acid is described in WO92/06216.
In previous conventional methods in nucleic acid diagnostics the amplification and detection of nucleic acids was carried out in separate systems so that the products produced by amplification came into contact with the surroundings before the detection. This resulted in a high risk of cross-contaminations and false-positive results. In addition it was necessary to carry out several pipetting steps and at least one transfer step.
A combined amplification and detection method in a closed system is known from U.S. Pat. No. 5,210,015 in which the amplification step is carried out in the presence of a detection probe. This detection probe binds under the amplification conditions to the target DNA and contains a reporter and a quencher group. The nuclease activity of the polymerase allows the reporter group to be cleaved from the probe generating a detectable signal. However, this method has the disadvantage of a low sensitivity. In addition the probe must be very carefully selected with regard to its sequence and melting point.
The publication Findlay et al. (Clin. Chem. 39 (1993), 1927-1933) discloses an automated method for the amplification and detection of nucleic acids in which a single reaction vessel with several separate compartments is used wherein the product mixture produced during the amplification in a first compartment has to be transferred into a second separate detection compartment for the detection. A disadvantage of this method is that reaction vessels with a complex construction or/and complicated transfer techniques have to be used.
WO93/10267 describes a method for the amplification and detection of nucleic acids in which a detection probe is present during the reaction which carries an electrochemiluminescent marker group at least at its 3′ end so that it cannot serve as a primer for the amplification. A disadvantage of this method is that a complicated construction of detection probes is necessary. Moreover it is not possible to exclude an interference of the amplification reaction by the added detection probe.
EP-A-0 628 568 discloses a method for the amplification and subsequent immobilization or detection of nucleic acids by hybridizing the nucleic acid with a capture probe in which the capture probe is protected against enzymatic extension or/and enzymatic degradation of the hybrid formed. The capture probe can optionally already be present during the amplification reaction. This method also has disadvantages in that the preparation of the protected capture probes is complicated and despite this it is not possible to exclude an interference of the amplification reaction by the capture probe.
REFERENCES:
patent: 5599668 (1997-02-01), Stimpson et al.
patent: 5750337 (1998-05-01), Squirrell
patent: WO 94/02634 (1994-02-01), None
Hlguchi et al., “Simultaneous Amplification and Detection of Specific DNA sequences”, Biotechnology, vol. 10, pp., 413-417, Apr. 1992.
Kleiber Jörg
Sagner Gregor
Tabiti Karim
Arent Fox Plotkin Kintner Kahn PLLC.
Chakrabarti Arun K.
Jones W. Gary
Roche Diagnostics GmbH
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