Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-01
2003-08-19
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007600, C435S320100, C435S196000, C536S023100, C536S023200, C536S024100
Reexamination Certificate
active
06607886
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method and apparatus for simultaneous quantification of the amounts of one or more radioactive nuclides within arbitrary regions on a surface where these nuclides have been deposited, adsorbed or fixed. These radioactive nuclides serve as markers on compounds that typically have been incorporated into tissue sections or into larger biological molecules that by various mechanisms have been bound to chemical substances on this surface. The method is especially well suited for DNA microarray deductions through the use of nucleotides labelled with different beta-emitting radionuclides.
BACKGROUND
It is widely believed that thousands of genes and their products, i.e. RNA and proteins, in a living organism function in a complicated and orchestrated way that creates the mystery of life. However, traditional methods in molecular biology generally work on a “one gene in one experiment” basis, which makes it hard to achieve the overall picture of the gene function. Thus, biological microarrays represents one of the most potent new tools in biological research to emerge in recent years, since it gives the opportunity to study a complete set of genes and their products simultaneously.
A microarray is an ordered arrangement of biological molecules immobilised in sample spots on a test plate which provides a medium for matching known and unknown samples of biological molecules. The immobilised molecules on the test plate are often denoted probe molecules, while the biological molecules from the test samples are denoted target molecules. In the case when the probe molecules and target molecules forms specific complementary pairs of biological molecules, the ordered arrangement of the test spots can be employed to identify specific biological molecules in a test sample from an organism and also to determine the abundance of these molecules. Typical examples of complementary biological molecules are hybridisation pairs of DNA, gene-anti gene etc. In microarrays the sample spots are typically less than 200 &mgr;m in diameter, but there “macroarrays” with sample spots with diameters of typically 300 &mgr;m or larger have been described.
The biological microarray technique can be applied for numerous applications such as diagnosis, identification/discovery of new genes and proteins, drug discovery, pharmacological and toxicological research etc. The technique can also be employed to comparison tests where biological components from several sources are adsorbed onto the same array, for instance from a healthy cell and a tumour cell.
Among biological microarrays, it is especially one type that has drawn attention the latest years; the DNA-microarray. This technology promises to monitor the whole genome on a single chip, and thereby make it possible to acquire a better picture of the interactions between thousands of genes simultaneously.
PRIOR ART
In general, the conventional method of determining biological activity by DNA-microarrays can be described as follows; strands of probe cDNA (typically 500-5000 bases long) are immobilised in a specific and ordered array onto a solid plate (typically a glass plate). Then the probe cDNA is exposed to one or several targets (marked cDNA from the test samples) either separately or in a mixture. The targets, labelled cDNA, are produced enzymatically by reverse transcriptase from samples of RNA which are extracted from the test samples and labelled with specific marker molecules. The reverse-transcribed RNA transcripts of the samples are hybridised with the probe cDNA on the microarray. Thus the amount and type of each target cDNA can be determined by measuring the location and concentration of each marker molecule at each test spot on the microarray, since the marker signal from each test spot reflects the relative transcript amounts for each specific transcript at each test spot on the microarray. To eliminate sample variation, the signal ratio between two competing samples is the preferred measurement.
Fluorescence Tagged Nucleotides
Traditionally, the detection of signals using this technology has been based on in vitro incorporated nucleotides labelled with suitable fluorophores, that is, specific fluorophores (fluorescence molecules) of a distinctive colour are inserted into the RNA extracted from each sample, respectively. Thus nucleotides labelled with fluorophores of a distinctive colour are incorporated into the target cDNA which will be hybridised to the probe cDNA. The fluorophores may be excited by different wavelengths, and similarly also emit at different wavelengths. Laser light and the use of appropriate filters to separate signals from two or more cDNA populations will generally achieve this.
However, the general need for starting material is in the range of 50 &mgr;g of total RNA, or approximately 5×10
7
cell equivalents. This relatively high amount of material excludes the use of standard technology from a number of very relevant applications, including clinical diagnostics. One highly significant factor in this lack of sensitivity is the low incorporation rate of current fluorophore-tagged nucleotides in the reverse transcription of cDNA from RNA. Thus only a relatively low number of fluorescence molecules will be incorporated per synthesised cDNA. Also, applications requiring incorporation of fluorescence through cell culture will be excluded, as fluorophore-tagged nucleotides will generally not be included through the cellular machinery. Emerging techniques to achieve better signal strength include enzymatic amplification of sample material and chemical signal amplification. Such methods demonstrate that it is possible to reduce sample size.
Amplification techniques have recently been published allowing a reduction in sample size down to 100 ng total RNA starting material (Wang, E. et al.
Marincola FM Nat Biotechnol
2000.18(4):457-459). This is achieved through one or more rounds of cycling between RNA and cDNA. In this reaction it is possible in each round to obtain approximately a 50-fold increase of the material by attaching a T7 promoter at one end to enable generation of RNA, and at the other end exploiting a feature of certain reverse transcriptases to add a specific primer to all cDNAs at most 5′ end at the time of first reverse transcription. This feature is necessary to avoid generation of shorter length cDNAs.
An alternative strategy is to amplify the signal from the test material through chemical means. The most sensitive strategy so far available relies on nested tangles of labelled branched synthetic DNA molecules (Nilsen, T. W. et al.
J. Theor. Biol.
1997.187:273-284; Wang, J. et al.
Electroanalysis
1998.10:553-556; Wang, J. et al.
J. Am. Chem. Soc.
1998.120:8281-8282). These may be bound to poly-A tails of cDNA prior to array hybridisation. Generally, a 250-fold increase in signal strength may be achieved.
Neither of these strategies have been rigorously tested for reliable performance and sensitivity levels. It is likely that amplification techniques will lead to degrees of bias of the starting material, due to the enzymatic nature of the process combined with the large variation in mRNA length for different transcripts.
The large amount of test material necessary to achieve adequate signal strength, and the problem that the available fluorophores are accepted only with difficulty by the reverse transcriptase enzyme represents thus considerable disadvantages in the prior art.
Radioactivity Labelled Nucleotides
It is known that the problem with low acceptance by the reverse transcriptase enzyme can be solved by employing radioactive isotopes for the labelling of nucleotides.
Historically, radioactive isotopes have been in widespread use for sensitive detection and quantification of nucleic acids. The common use has been confined to the use of one single, usually beta-emitting, radionuclide, incorporated either into a probe (detector nucleic acid) or directly in vivo. The detection has been performed using liquid scintillation or gamma counters, and in
Breistol Knut
Hovig Eivind
Kvinnsland Yngve
Nygard Einar
Skretting Arne
Biomolex AS
Chakrabarti Arun Kr.
Jones W. Gary
Licata & Tyrrell P.C.
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