Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1985-10-15
1989-06-20
Warden, Robert J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 7, 435 18, 435 25, 435 26, 435 28, 435810, 435817, 436501, 436537, 436806, 436904, 536 26, 536 27, 536 28, 935 77, 935 78, C12P 168, G01N 3353, G01N 33566, C07H 1906
Patent
active
048408930
DESCRIPTION:
BRIEF SUMMARY
The present invention is concerned with assays for nucleic acids, the said assays extending to both detection of the presence f nucleic acids and to the particular identification of specific nucleic acid molecules.
There is a particular need in many areas of biochemical research and commerce to be able to assay for the presence of nucleic acids in a sample, and further, to assay for the existence of particular sequences within the said nucleic acids.
The exploitation of the hybridisation between two single-stranded DNA or RNA molecules which have a complementary sequence has been one approach to this problem. Molecules for use in such assays, which are capable of binding to a DNA or RNA sequence which is defined to a particular level of homology, are known as DNA-probes.
Known DNA (RNA) probe techniques share a similarity in that the DNA (RNA) polymer is not readily detectable by its inherent biochemical activity. It is therefore necessary to mark the polymer with some signal-producing chemical or biochemical species, such methods as are presently known include the followin prior art:
PRIOR ART
Avidin-Biotin Reaction; this technology relies on the affinity of the egg-white glycoprotein avidin for biotin. Biotin (Vitamin H) can be covalently linked to the nucleotide residues which comprise the monomeric subunits of the DNA polymer. The modified subunits can still undergo the classical binding reaction between complementary strands of double-stranded DNA, and thus can be incorporated into synthetic DNA probes. At present, to detect the presence of such probes which have formed short double-stranded regions after exposure to complementary sample DNA, the unbound probe must first be separated from the sample DNA/bound probe complex. This is normally done by performing the binding reaction in conditions under which the sample DNA is immobilized on a substrate and washing, although centrifugation may perform the same function. The bound probe is detected by the addition of avidin to which either a fluorescent- marker-labeled antibody or an enzyme has been attached.
One problem with the above method is that small oligonucleotide probes (20 nucleotides) contain only a small number of biotinylated sites, limiting the amount of avidin which can be bound. Attempts have been made, with some success, to add long "tails" of up to several thousand bases to the probe DNA, in which case only the tail need be labeled. The method can detect up to a resolution of 10.sup.-13 g of DNA, or about 10.sup.5 copies of a single gene. Although originally the marker on the avidin was horse radish peroxidase the method has now been extended to include alkaline phosphatase. Unfortunately the method is generally difficult to establish for a new diagnostic scheme as either the biotin-linked probe DNA is difficult to prepare or the labeled tail interferes with sensitivity.
Use of Mediators
Commonly owned European Patent Application No. 84303090.9 published on Nov. 14, 1984 as EP No. 125139A discloses a method in which at least one of a mediator and an enzyme are chemically linked to a nucleic acid probe sequence whereby specific binding of the probe sequence to the target sequence in a single-strand nucleic acid material to be investigated affects the electrochemical availability of the chemically linked species as detected by a sensor electrode in presence of the enzyme substrate, whereby the presence of the target sequence can be detected.
As disclosed in the specification, the nucleic acid sequence can be RNA e.g. messenger RNA but is usually DNA.
A suitable method for the practice of this technique has been given as follows; a given target sequence; complementary to the target sequence; enzyme-linked probe to a solution containing both a substrate for the enzyme and a mediator, mediator-linked probe to a solution containing both a substrate and an enzyme for the said substrate; adding the so-modified probe to a solution containing a substrate for the enzyme; with a sensor electrode whereby charge is transferred by the mediator to
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Hill Hugh A. O.
Libor Susan I.
MediSense, Inc.
Spiegel Jack
Warden Robert J.
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