Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-30
2001-12-11
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024320
Reexamination Certificate
active
06329152
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The field of this invention is cellular RNA detection. More particularly, the present invention pertains to a process for detecting the presence of a specific target mRNA molecule present in low abundance in intact cells.
BACKGROUND OF THE INVENTION
Many approaches have been used to identify nucleic acids within cells and tissues. In situ PCR (ISPCR) is extremely sensitive as this technique has been shown to detect single copy DNA and low abundance RNA. ISPCR, however, yields no information on the starting target copy number and thermal amplification of cells increases cellular autofluorescence five-fold. Conversely, in situ hybridization allows quantification of the starting copy number though the sensitivity ranges between 100-1000 copies. Further, the current art of in situ hybridization uses compounds such as dextran sulfate, acetic anhydride, polyethylene glycol (PEG), hydrochloric acid, and others that either fluoresce or increase cellular autofluorescence. In sum, compounds that either fluoresce in the range of the reporter fluorescent dye or increase cellular autofluorescence decrease signal to noise (SNR) and sensitivity. The present invention provides a method to detect and quantify intracellular nucleic acids with a sensitivity between 3-100 copies. This level of sensitivity is acheived by using a novel combination of non- or weakly crosslinking fixatives and exclusion of autofluorescent compounds commonly used for in situ hybridization. In addition, this method allows simultaneous analysis of cell surface markers including but not limited to phenotypic markers, activation markers, functional markers, and antigens associated with cell death injury.
The optimal detection system should be able to detect a very few copies of a particular target with a broad, linear range for quantification. In addition, this detection scheme should allow simultaneous multiparameter (immunophenotypic) analysis and should be adaptable for use on multiple detection platforms (flow cytometer, image analysis). Last, this optimal test should be easy to perform with high throughput capabilities. The most important determinants of successful in situ hybridization experiments are access to target and signal to noise ratio (SNR). Access to intracellular targets, whether protein or nucleic acids, has always been a challenge. In addition, proteins bound to nucleic acids provide additional obstacles for in situ detection. The approaches to overcome these obstacles depend on the cells or tissue. Cells in suspension or adhered to slides are generally intact. Access to nucleic acids in cells involves permeabilization of the cell membrane and removal of protein bound to nucleic acids. Many agents have been used to permeabilize and many have been commercialized as “fix and perm” combinations. In the past, methanol was used to extract lipids, protease were used to digest membrane associated proteins, and saponin was used to extract membrane associated cholesterol. Methanol, however, was a poor fixative and protease treatment was temperamental with a fine line between optimal use and complete obliteration of cells, and saponin was required in all solutions following the fixation step to maintain permeability.
The classic model systems illustrating sensitivity (high SNR) of detection schemes are human papilloma virus infection and HIV infection. The human papilloma virus (HPV) infected cell lines, SiHa and Caski, contain different number of HPV copies. SiHa cells contain two copies of HPV DNA and Caski cells contain about 300 copies of HOV DNA. In situ hybridization can detect HPV DNA in Caski cells but not SiHa cells. In situ PCR, on the other hand, can detect HPV DNA in both cell lines. In situ PCR, however, is inconsistent, technically difficult, and has a low throughput.
Similarly, the HIV life cycle in cells presents the ultimate challenge for gene detection. Determinants of viral replication including expression of unspliced HIV mRNA and plasma free virus has led to the use of virologic markers as a measure of disease status and therapeutic efficacy. A marked increase in the ratio of unspliced to spliced HIV mRNA, as might occur during the shift from latent to productive infection, precedes precipitous drops in CD4 count. Plasma viral load has been shown to correlate with disease progression and has been used to determine HIV kinetic in vivo. These measurements, however, fail to provide information on the cell type of origin, a weakness considering, the effect of HIV gene expression on cell function, the role of infected cells in transmission and dissemination, and the therapeutic potential of blocking cell-type specific coreceptors.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a process for detecting the presence of a target mRNA in an intact cell. The process includes the steps of (a) preserving and permeabilizing the cell with a non or weakly crosslinking fixative in the presence of a plurality of oligonucleotide probes, wherein each probe (i) contains about 6 to about 30 nucleotides, (ii) is labeled with a detectable marker, (iii) has a matched Tm of greater than about 60° C., and (iv) specifically hybridizes to a different contiguous region of an open reading frame in the target mRNA with the provisos that each probe does not hybridize to itself, does not hybridize to any other probe and does not hybridize to a contiguous sequence of (A)
n
(C)
n
(G)
n
or (U)
n
in the target mRNA where n is an integer greater than 5; (b) removing unhybridized probes from the cell; and (c) measuring the detectable label in the cell.
The process can be used to detect target mRNA in a cell in a copy number of from about 3 to about 100. More particularly, the process can be used to detect mRNA present in copy numbers of from about 3 to about 100, from about 3 to about 50 and more preferably in copy numbers from about 3 to about 25.
The process can be used to detect mRNA that is indigenous to the cell or present in the cell as a result of introduction from an outside source such as gene transformation or viral infection. In a preferred embodiment, the process is used to detect cellular mRNA such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The process can be used with any cell from any animal. The process is particularly suited for detection of mRNA in white blood cells of human patients. A preferred white blood cell is a lymphocyte such as a T-lymphocyte.
The detectable label used in accordance with the process is preferably a fluorescent label such as 6-carboxyfluorescein. Each of the plurality of oligonucleotide probes is labeled with the same fluorescent label or in another embodiment different fluorescent labels.
The present process can be used simultaneously with other processes such as a process for detecting the presence of an immunogenic or molecular marker of cell function in the cell. In preferred embodiments, the marker is a marker of cell phenotype, cell activation, or cell death.
REFERENCES:
Patterson BK et al. Detection of HIV-1 DNA and mRNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science, 260: 976-979, 1993.*
Mosiman et al. Reducing cellular Autofluorescence in flow cytometry: an in situ method. Cytometry, 30: 151-156, 1997.*
Hougaard DM et al. Non-radioactive in situ hybridization for mRNA with emphasis on the use of oligodeoxynucleotide probes. Histochem Cell Biol., 108: 335-344, 1997.
Campbell Eggerton A.
Chundru Suryprabha
Niro Scavone Haller & Niro
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