Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-14
2002-07-23
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S199000, C435S252300, C435S091210, C435S091200, C536S022100, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06423492
ABSTRACT:
TECHNICAL FIELD
This invention relates to methods for detecting nucleic acids.
BACKGROUND ART
Nucleic acid hybridisation is a widely used technique for identifying, detecting and quantitating target polynucleotide sequences in a sample. This technique relies for its success on complementary base pairing between the two halves of a double-stranded nucleic acid molecule: when single-stranded nucleic acids are incubated in solution under suitable conditions of temperature, pH and ionic strength, complementary base sequences pair to form double-stranded stable hybrid molecules. This ability of single-stranded nucleic acid molecules to form a hydrogen-bonded structure with their complementary nucleic acid sequences has long been employed as an analytical tool in recombinant DNA research.
In most cases the sample will contain double-stranded nucleic acid and must be denatured prior to the hybridisation assay to render it single-stranded. A nucleic acid having a known sequence which is complementary to the target sequence is either synthesised chemically in an automated fashion with great facility, or is isolated from the appropriate organism and rendered single-stranded by denaturation. It is then used as a probe to search a sample for a target complementary sequence. Detection of specific target nucleic acids enables accurate diagnosis of bacterial, fungal and viral disease states in humans, animals and plants. Additionally, the ability to probe for a specific nucleotide sequence enables the diagnosis of human genetic disorders. Hybridisation produces stable hybrids, and a number of different approaches are known to the art for detecting these.
One approach involves the use of labelled probes. By labelling a probe nucleic acid with some readily detectable chemical group, it is possible to detect the polynucleotide sequence of interest in a test medium containing sample nucleic acids in single-stranded form. Nucleic acids have been labelled with radioisotopes, enzymes and fluorescent molecules. The use of labelled nucleic acids as probes in macromolecuiar analysis is important for clinical, veterinary and environmental diagnostic applications.
Early methods for detecting target nucleic acids involved their immobilisation on a solid support such as nitro-cellulose paper, cellulose paper, diazotized paper, or a nylon membrane. For example, in U.S. Pat. No. 4,358,535 to Falkow a method is disclosed in which the target nucleic acid is rendered single-stranded and then immobilised onto a membrane. A labelled probe which is complementary to the target nucleic acid is brought into contact with the solid support and hybridises to the target nucleic acid. The solid support is washed several times at a carefully controlled temperature to remove unbound and non-specifically bound probe without removing specifically bound probe, and the presence of the label in the resulting hybrid is determined. A disadvantage of this method is that it is neither easy nor convenient to attach the single-stranded target nucleic acid to a solid support, the whole process involving a 12-15 hour incubation of the nucleic acid with a nitro-cellulose sheet, followed by a 2 hour baking step. This makes the assay slow and unattractive for routine use. It is also cumbersome, with the hybridisation and washing steps being carried out in a sealed pouch, containing the membrane and the buffer solution. In addition, when very low concentrations must be detected, the ratio of specific to non-specificaily bound probe can be very low and repeated washing under highly stringent conditions is frequently required. Under these conditions the sensitivity of the assay is often compromised because of substantial loss of specifically bound probe.
Since then a many improvements have been made, most of which employ a sandwich approach using two probes: a reporter probe and a capture probe. The reporter probe is a nucleic acid having a sequence complementary to at least part of the target sequence and which is labelled with a detectable group. The capture probe is a nucleic acid having a sequence complementary to at least part of the target sequence, but which is different to that of the reporter probe, and which is labelled with an immobilisable group. In many applications, pairs of specific binding members (sbm's) have been used for this purpose.
For example, in U.S. Pat. No. 5,273,882 to Snitman and Stroupe a capture probe complementary to part of the target nucleic acid is labelled with an antigen or antibody. After hybridisation between this capture probe and the target, the solution is introduced to a support-bound antibody or antigen which immobilises the hybrid formed between the capture probe and the target. Following a washing step, a second, reporter probe, complementary to a different region of the target nucleic acid, is introduced and the triple sandwich formed is detected.
Similar approaches are described by Holtke et al.: in U.S. Pat. No. 5,344,757 is disclosed a method in which a reporter probe is labelled with digoxin or digoxygenin, and hybrids are captured using antibodies against this hapten. In this case, a capture probe is not used, and the method is limited either to the detection of an immobilised target, or when the assay is used for detecting PCR products, one of the primers is immobilised. In U.S. Pat. No. 5,354,657 the method is further developed and involves the solution hybridisation between the target nucleic acid and a reporter probe labelled with digoxin or digoxygenin. This hybrid is captured by a solid-supported capture probe, complementary to a different region of the target. A detectably labelled antibody against the hapten is then added and the hybrids formed detected.
Specific binding members other than antigens or haptens and antibodies have been used. In U.S. Pat. No. 5,374,524 to Miller is described a method for the solution sandwich hybridisation, capture and detection of amplified nucleic acids. Amplicons are denatured and treated with an enzyme-labelled reporter probe and a biotinylated capture probe. Hybrids formed are captured using streptavidin-coated chromium dioxide particles.
Disadvantages of these approaches include the increased cost and complexity of using two probes. For example, for each assay two probes need to be synthesised and labelled: one for use as the capture probe, and the other for use as a reporter probe. In addition, hybridisation conditions have to be carefully chosen to form the sandwich of target, capture probe and reporter probe.
Simpler approaches which avoid the use of a capture probe have been described. Atlas and Steffan (
Biotechniques
(1990) 8:316-318) disclose a solution hybridisation method for detecting genetically-engineered micro-organisms in environmental samples. The detection method involves recovery of DNA from the microbial community of an environmental sample followed by hybridisation in solution with a radio-labelled RNA gene probe. After nuclease digestion of non-hybridised probe RNA, the DNA-RNA hybrids formed in the solution hybridisation are separated by column chromatography and detected by liquid scintillation counting. A less cumbersome approach is disclosed in U.S. Pat. No. 4,978,608 to Kung and Nagainis in which DNA is detected in a sequence non-specific manner using a high affinity single-stranded DNA-binding protein. This approach is extended in U.S. Pat. No. 5,536,648 to Kemp et al. who disclose an amplified DNA assay using a double stranded DNA binding protein. The method uses a PCR primer having a nucieotide sequence which is a ligand for a double stranded DNA-binding protein. After amplification the amplified target is captured by the double stranded DNA-binding protein immobilised on a solid surface. This method does not use a capture probe and will detect any amplification product containing the sequence which is a ligand for the double stranded DNA-binding protein. A disadvantage of this approach is that it relies an the accuracy of the amplification step for its specificity.
Another method is disclosed in U.S. Pat. No. 4,
Hopgood, Calimafde Judlowe & Mondolino
Wilder Cynthia B.
Zetatronics Limited
Zitomer Stephanie W.
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