Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-01
2001-02-13
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S005000
Reexamination Certificate
active
06187567
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to amplification and detection of target nucleic acid molecules. More particularly, the present invention relates to methods and reagents for retaining amplified target nucleic acid molecules within a cell upon amplification in situ, as well as for detecting the amplified molecules. The present invention thus improves the amplification and detection of a target nucleic acid molecule in situ.
In situ amplification and detection of nucleic acid molecules is useful for diagnosis, prognosis, drug efficacy determination, tissue pathology evaluation, patient monitoring, and many other clinical and research applications.
BACKGROUND OF THE INVENTION
Methods have been developed to study various aspects of gene expression to answer questions relating to, for example, how cells fulfill their various functions, how pathogens affect host cells, and how cells are transformed to a malignant state. In one method, RNA molecules may be isolated, amplified in a liquid phase, and then detected. However, this method does not reveal which cells are producing the RNA molecule of interest. In some instances, it is important to know what percentage of a cell population, or subset of a cell population, is expressing particular RNA molecules.
For instance, in most herpes virus infections, clinical disease corresponds with the production of certain viral RNAs in active infections, while not corresponding with the production of other viral RNAs or viral DNA that is produced during latent infection. As another example, HHV-8, which is thought to be the causative agent of Kaposi's sarcoma, has been found in Kaposi's sarcoma biopsies and peripheral blood samples. In situ studies indicate that viral infection is targeted to the endothelial (spindle) cell component thought to be the central cell in the pathogenesis of the lesion. Certain transcripts are thought to be indicative of viral replication, and detection of these transcripts may be able to distinguish latent from active infections (Zhong et al. (PNAS 93:6641(1996)).
In situ amplification may also be used for detecting human papilloma virus (HPV) infections and for determining the role the virus plays during transformation of cervical cells (See U.S. Pat. No. 5,506,105). Certain HPV transcripts are produced only in cells actually transformed to the malignant state, and the detection of these transcripts therefore corresponds specifically with the detection of early malignancy.
Nucleic acid amplification has also been used advantageously for detecting HIV-1 exposure in seronegative sexual partners of HIV-1 seropositive individuals, in HIV-1 seronegative infants and children, and in health care workers accidentally exposed to HIV-1 positive blood or body fluids. The ability to identify individual cells, either latently or productively infected, under the microscope would be very useful for delineating a latent state and emergence from it. This would be useful not only for understanding the development of infection, but also as a more direct quantitative measure of the effect of antiviral therapy. Further, the ability to identify individual infected cells would help understand the mechanism of HIV-1 transmission.
Thus, there exists a substantial interest in detecting in cells and tissues the distribution and/or expression of RNA molecules. In situ amplification of RNA targets, for example, provides a powerful technique to differentiate neighboring cells in a histochemical or cytochemical preparation with respect to somatic mutation, pathogenic infection, oncogenic transformation, immune competence and specificity, state of differentiation, developmental origin, genetic mosaicism, cytokine expression, and other characteristics useful for understanding both normal and pathological conditions in eukaryotic organisms.
In some instances, levels of target nucleic acids are sufficiently high such that they can be visualized with in situ hybridization using a labeled probe. However, at least one limitation inherent in such techniques is the copy number of the target molecule; the detection of RNA molecules by hybridization typically requires tens to hundreds of copies of the target nucleic acid molecule per cell. In many instances, the target nucleic acid molecules of interest are present only at low levels, in which case other techniques must be used.
PCR has been employed to amplify low copy number nucleic acids so that sufficient amounts of target sequence are available for hybridization and subsequent visualization (Haase et al., PNAS 87:4971, 1990). While this technique overcomes the problem of low copy number, methods involving thermal cycling may have a deleterious effect on the structure of cells and tissues. For instance, it may be difficult after thermal cycling to identify or classify the cells that contain the desired transcript, thus reducing the advantages and useful information gained from using the PCR method.
The sensitivity of PCR amplification has been combined with the target localization of in situ hybridization to create in situ PCR, wherein PCR is performed within chemically fixed cells (Haase et al 1990, PNAS 87:4971). Haase et al. used a series of overlapping primer pairs specific to overlapping target sequences within the genome of selected organisms to improve the retention of amplified target nucleic acids within cellular compartments.
One variation of conventional PCR is RT/PCR, which detects target RNA sequences in test samples via complementary DNA (cDNA) sequences reverse transcribed from the target RNA; the cDNA is then detected using conventional PCR (Kawasaki et al., 1988, PNAS 85(15):5698, and Rappolee et al., 1989, J. Cell. Biochem. 39:1-11). However, prior methods utilizing RT/PCR also have certain limitations, including, in addition to its deleterious effect on morphology, RT/PCR requires that only RNA be amplified in a background of DNA. In vitro RT/PCR methods solve this problem in two general ways: 1) DNase treatment, and 2) differentiation of the RNA amplification product from the DNA amplification product. Option 1 is more difficult to perform in in situ preparations than in reactions in solution. Option 2 usually requires that the two possible amplification products have different sizes and be resolved differently on a gel. Option 2 also requires that the two possible products hybridize to different sequences and differently labeled probes, thus also limiting this option in in situ methods.
In situ amplification methods also suffer from the problem that the amplification products, being small molecules, may diffuse from the cell as a result of permeations made in the cell surface to allow amplification reagents to enter the cell. There thus exists a need to prevent this phenomenon, called leakage, from occurring. In situ amplification methods thus demand a balance between, first, permeabilizing sufficiently the cell membrane to allow the amplification reagents to reach the target nucleic acid, and second, maintaining sufficient integrity in the membrane to prevent leakage of the amplifed target material and enable detection.
Methods for solving the leakage problem during in situ PCR amplification have used multiple primer sets. Such methods produce overlapping amplification products of different sizes, wherein the products are partially double-stranded, each strand having overhanging ends that can hybridize to one another to form large concatamers and retard diffusion from the cell. However, in addition to suffering from the above drawbacks associated with standard in situ PCR (e.g., poor morphology due to the thermal cycling), the use of multiple primer pairs to solve the leakage problem has additional disadvantages, including the increased expense for their production and the inherent problem that with many target organisms, such as pathogenic viruses, it is hard to find even a few short sequences that will make good primer and probe sites. The use of multiple primer pairs also has the disadvantage of increasing the likelihood of non-specific background
Hickman Kathy Lynn
Hurteau Gregory John
Lee Eun Mi
Li Yuexia
Akzo Nobel N.V.
Einsmann Juliet C.
Fredman Jeffrey
Sullivan Michael G.
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