Polynucleotide probe and kit for amplifying signal detection...

Chemistry: analytical and immunological testing – Process or composition for determination of physical state... – Corrosion resistance or power

Reexamination Certificate

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C435S006120, C536S024300, C530S387100

Reexamination Certificate

active

06306657

ABSTRACT:

BACKGROUND
The present invention relates to nucleic acid hybridization assays which are useful as a means of locating specific nucleic acid sequences. Examples of nucleic acid sequences are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) sequences. The molecular subunits of both DNA and RNA are called nucleotides which are linked together to form long polynucleotide chains. Each nucleotide subunit is made of a sugar moiety, a phosphate moiety and a base moiety. It is the sequential ordering of the base moieties [adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)]that contains DNA or RNA's genetic information. The ordering of these base moieties in a polynucleotide chain and the tendency of the bases to attract and bond with other specific base moieties, is exploited by this invention to locate, detect and isolate specific DNA or RNA sequences.
DNA normally contains two polynucleotide strands twisted about one another lengthwise in a helical manner resembling a ladder where the sides are made of identical sugar (deoxyribose) and phosphate molecules while the rungs are made up of bases extending out from each strand, held together by weak attractive forces. In DNA, the base thymine on one strand always pairs with the base adenine on the opposing strand, and the base guanine always pairs with the base cytosine. This is called complementary base pairing.
RNA is also a polynucleotide strand. However, the sugar moiety is ribose (versus deoxyribose in DNA) and the bases are adenine, guanine, cytosine and uracil. In RNA, the base uracil on one strand always pairs with the base adenine on the opposing strand, and the base quanine always pairs with the base cytosine. Although RNA can pair with either a complementary strand of RNA or DNA, it is normally single stranded so does not form a helical structure.
The present invention is founded, in part, upon the technique that single stranded nucleic acid sequences can be combined, or hybridized, under appropriate conditions with complementary single stranded nucleic acid sequences to form double stranded molecules. This technique was developed as a means for detecting and/or and isolating particular nucleic acid sequences of interest. It has increased in popularity during recent years in its application for detecting the presence of the DNA or RNA within such pathogens as viruses, bacteria, or other microorganisms and therefore the presence of these pathogens themselves. The technique can also be used for other purposes such as to screen bacteria for antibiotic resistance, to aid in the diagnosis of genetic disorders (for example in sickle cell anaemia and thalassaemia), and to detect cancerous cells. Several applications have been developed for the microbiological analysis of clinical, food, environmental, and forensic samples. A general review of the technique and its present and future significance is provided in Biotechnology (August 1983), pp. 471-478 which is incorporated herein by reference.
The following definitions are provided to facilitate an understanding of the present invention. The term ‘probe’ refers to a nucleic acid sequence of which there are at least three types: the primary probe, the amplification probe, and the labelling probe. The primary probe contains at least one nucleic acid sequence that is complementary (or will base pair) to some portion of a nucleic acid sequence on the target DNA or RNA molecule of interest. The amplification probe contains sequences that are complementary to some sequences on the primary probe, and contains a region that is typically of at least one type of repeating sequence unit. The labelling probe contains sequences complementary to one of the repeating sequence units, in addition to a chemical label. Labels are detectable chemical groups, either radioactive molecules or non-radioactive molecules and can include: radioactive isotopes; enzymatically active groups such as horse radish peroxidase; fluorescent agents; chemiluminescent agents; precipitating agents; and/or dyes. The term ‘signal’ is used loosely to indicate the detectable characteristic of a detectable chemical group, which can include: a change in the light adsorption characteristics of a reaction solution resulting from enzymatic action of an enzyme attached to a labelling probe acting on a substrate; the color or change in color of a dye; fluorescence; phosphorescence; radioactivity; or any other indicia that will be evident to one skilled in the art.
The amplification probe is so named because it is used to cause many detectable chemical labels to become attached to one probe-target complex, such that the resulting signal is amplified in direct proportion to the number of labelled probes that hybridize to the amplification probe. If the amplification probe were to contain only one sequence unit that comprises sequences compatible to the labelling probe, only one labelling probe would become attached to the probe-target complex, and the signal would not be amplified. However, the amplification probe disclosed in the present invention contains typically five or more sequence units (also referred to as repeatmers) that are compatible to the labelling probe, such that five labelling probes will attach to one probe-target complex, resulting in five times the amount of detectable chemical label signalling the presence of one probe-target complex; thus, the indication that one probe-target complex was formed will be amplified five times. Moreover, if the amplification probe contains sixteen sequence units that are combatable to the labelling probe, sixteen labelling probes will attach to one probe-target complex, resulting in sixteen times the amount of detectable chemical label signalling the presence of one probe-target complex, the indication that one probe-target complex was formed will thereby be amplified sixteen times. The degree of amplification is optional and can be manipulated by the design and construction of the amplification probe as described herein.
One objective of a nucleic acid hybridization assay is to detect the presence of a specific nucleic acid sequence (the target sequence) in a given sample by contacting the sample with a complementary nucleic acid sequence (the probe) under hybridising conditions and observing the formation or absence of any probe-target complexes. The probe-target complex can be detected directly by a label attached to the probe. The complex can also be detected indirectly through such techniques as the hybridization of another nucleic acid sequence conjugated to a label or by the binding of an antibody labelled with a detectable chemical group.
One detection strategy currently employed in the art is exemplified by PCT Application 84/03520 and EPA 124221 which use an enzyme labelled nucleic acid sequence to detect the probe-target complex by hybridization to complementary sequences on the tail of the probe. For example, the Enzo Biochem “Bio-Bridge” system uses a biotin molecule conjugated to a poly(A) tail (a nucleic acid sequence comprised solely of adenine nucleotides) as the detection system following hybridization of a DNA probe possessing a poly(T) tail (a nucleic acid sequence comprised solely of thymine nucleotides) to the target DNA sequence.
In order to employ such a technique as an assay, one must be able to detect the presence or absence of probe-target complexes with a high degree of sensitivity. The sensitivity of a nucleic acid hybridization assay is determined primarily by the detection limit of the label to demonstrate the formation of the probe-target complex against back-ground noise and/or false-positives. Different strategies have been employed to improve the sensitivity of nucleic acid hybridization assays, which can be classified into four broad categories: 1) separation of the probe-target complex; 2) target amplification; 3) probe amplification; 4) multiple labelling, or combinations thereof.
Some nucleic acid hybridization assays involve immobilization of the target sequence on a solid support followed by washing away the re

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