Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1988-06-16
1993-10-26
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
436 82, 436546, C12Q 168, G01N 3320, G01N 33533
Patent
active
052565351
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to a hybridization assay utilizing nucleic acid probes labelled with lanthanide chelates that show time-delayed fluorescence.
Labelled nucleic acids have become indispensable in hybridization assays, performed both in vitro and, as in hybridocytochemical microscopy, also in vivo. Appropriate labelling of nucleic acids is a crucial point in their sequencing and applications may also be found in the different methodologies of nucleic acid separation. Nowadays, most of the efforts to find more sensitive markers have been made in the field of hybridization probes for the detection of specific, complementary nucleic acid sequences. This is natural in view of the great importance of such assays in medicine and molecular biology.
DNA or RNA can be labelled in a variety of ways. Generally, all labels may be detected directly i.e. the label, which is bonded to the nucleic acid is itself detectable, or indirectly when the label participates in one or more reactions thus generating detectable products.
DESCRIPTION OF PRIOR ART
Several new labelling methodologies have recently become available. Despite of many differences they can be systematized on the basis of some main criteria. In nucleic acid technology a common name for different labels is reporter group.
1. Methods of DNA Detection
a) Direct detection method: Nucleic acids are commonly labelled with the radioisotopes .sup.32 P, .sup.125 I, .sup.3 H or fluorescent markers. Especially in routine analyses the radioactive material tends to be replaced by the non-radioactive labels because of the serious drawbacks associated with the use of radioactive labels. Safety and disposal problems are obvious, but the low stability of materials with high specific activity together with their high cost should not be forgotten either.
Theoretically, fluorescent compounds are ideally suited to replace radioactive isotopes. To date the only examples of such fluorescent markers used in DNA labelling are fluorescein, rhodamine, Texas Red and NBD. The calculated high sensitivity which could be achieved using this type of reagents is, however, to a great extent limited by the fact that most biological samples including proteins also fluoresce thus bringing the background to a not always acceptable level to distinguish between the fluorescent marker and the protein.
b) Indirect detection method: Nucleic acids are labelled with different proteins possessing enzymatic activities e.g. alkaline phosphatase or horse radish peroxidase. The subsequent reaction of the appropriate substrate catalyzed by the attached enzyme usually generates an easy-to-detect colored product. It is constantly emphasized that an advantage of such a system is the fact that there is no need for a detecting apparatus, but this fact can also be seen as a disadvantage since this visual technique is not well-suited to quantitative analysis.
2.Attachment of a Detectable Group to DNA or RNA
The way in which report groups are linked to nucleic acids may serve as another criteria of differentiating between labelling techniques.
Direct attachment means that a detectable marker is bound to a nucleic acid already before a particular analytical process takes place. The label may be coupled to the nucleic acid enzymatically as in radiolabelling, by using DNA kinase and .tau. .sup.32 P ATP, or by employing different .sigma. .sup.32 P nucleoside triphosphates in the nick translation process. A properly activated label which is able to react with any existing or created function in nucleic acids, can also be chemically attached to nucleic acids.
Indirect attachment of the detectable group to nucleic acids can be realized by several methods. Among the most commonly used methods is the labelling of nucleic acids with a hapten, thus rendering them detectable by immunological means.
Examples of haptens useful in indirect attachment are fluorescein and a trinitrophenyl group (TNP). Antibodies with high affinity to these haptens have been well-studied and are easily obtainab
REFERENCES:
patent: 4259313 (1981-03-01), Frank I
patent: 4283382 (1981-08-01), Frank II
patent: 4670379 (1987-06-01), Miller
patent: 4707440 (1987-11-01), Stravianopoulos
patent: 4795700 (1989-01-01), Dervan et al.
patent: 4801504 (1989-01-01), Burdick
patent: 4996142 (1991-02-01), Al-Hakim et al.
Richardson "Terbium (III) and Europium (III) Ions as Luminescent Probes" Chem. Rev. 1982 vol. 82, pp. 541-552.
Nucleic Acid Research vol. 2, pp. 1017-1028, published 1986 (Syvanen A-C et al) "Time-resolved fluormetry: a sensitive method to quantify DNA-hybrids".
Dahlen Patrik
Hurskainen Pertti
Kwjatkowski Marek
Lovgren Timo
Sund Christian
Kim Hyosuk
Wax Robert A.
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