Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-11-02
2001-10-09
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S283100, C435S287100, C435S286100, C435S286200, C435S091200, C435S091100
Reexamination Certificate
active
06300124
ABSTRACT:
BACKGROUND OF THE INVENTION
The polymerase chain reaction (PCR) is a technique involving multiple cycles that results in the geometric amplification of specific polynucleotide sequences present in a test sample each time a cycle is completed. To amplify the specific nucleic acid sequences (“target sequences”), PCR reagents are combined with the test sample. These reagents include, for example, an aqueous buffer, pH 8-9 at room temperature, usually also containing approximately 0.05 M KCl; all four common nucleoside triphosphates (e.g., for DNA polymerase, the four common dNTPs: dATP, dTTP, dCTP, and dGTP) at concentrations of approximately 10
−5
M to 10
−3
M; a magnesium compound, usually MgCl
2
, generally at a concentration of about 1 to 5 mM; a polynucleotide polymerase, preferably a thermostable DNA polymerase, e.g., the DNA polymerase I from Thermus aquaticus, at a concentration of about 10
−10
to 10
−8
M; and single-stranded oligonucleotide primers, preferably deoxyribo-oligonucleotides, usually 15 to 30 nucleotides in length, containing base sequences which have Watson-Crick complementarity to sequences preferably on each strand of the target sequence(s). Each primer is present at a concentration of about 10
−7
to 10
−5
M.
Initially, a reaction tube containing the test sample is heated to a temperature at which nucleic acid sequences are denatured, generally 90° C. to 100° C. Then the sample is subjected to a temperature at which oligonucleotide primers, preferably at least two oligonucleotide primers, can anneal to opposing strands of the target sequence, generally 40° C. to 75° C. The polymerase then catalyzes the incorporation of nucleoside monophosphates, beginning at the 3′ end of the primer (“primer extension”), generally at 40° C. to 75° C.
The practical benefits of PCR nucleic acid amplification have been rapidly appreciated in the fields of genetics, molecular biology, cellular biology, clinical chemistry, forensic science, and analytical biochemistry. For example, see Erlich (ed.)
PCR Technology
, Stockton Press (New York) (1989); Erlich et al. (eds.),
Polymerase Chain Reaction
, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.) (1989); Innis et al.,
PCR Protocols
, Academic Press (New York) (1990); and White et al.,
Trends in Genetics
5/6: 185-189 (1989). PCR can replace a large fraction of molecular cloning and mutagenesis operations commonly performed in bacteria, having advantages of speed, simplicity, and lower cost. Furthermore, PCR permits the rapid and highly sensitive qualitative and even quantitative analysis of nucleic acid sequences.
Although one can move PCR reaction tubes manually back and forth between thermostated baths in each temperature range, PCR most commonly is performed in an automated temperature-controlled machine, known as a “thermal cycler,” in which a microprocessor is programmed to change the temperature of a heat-exchange block or bath containing reaction tubes back and forth among several specified temperatures for a specified number of cycles, holding at each temperature for a specified time, usually on the order of one-half to two minutes. The total cycle time is usually less than 10 minutes, and the total number of cycles is usually less than 40, so that a single, multi-cycle amplification, amplifying the targeted nucleic acid sequence 10
5
to 10
10
times, normally occurs in less than seven hours and often less than four hours.
PCR has also been applied to amplify specific DNA segments inside cells, without first extracting the DNA from the cells. This technique is called in situ PCR. The cells may be individual cells, or part of a tissue sample. Most often, in situ PCR is performed on cells or thin slices of tissue (“tissue sections”) mounted on microscope slides. Cells which do not form tissues, such as leukocytes and many cultured cells (such as HeLa cells), are spread out upon a slide by centrifugation, producing a “cytospin” preparation. The cells or tissue usually have been fixed by treatment with formalin, or other reagents (“fixatives”), so that their morphology is preserved and recognizable after PCR and subsequent detection of the amplified nucleic acid.
To perform in situ PCR on fixed cells or tissue samples on a glass microscope slide, the slide is pretreated with an agent that inhibits or prevents the cells or tissue from being removed during the PCR process, or during the subsequent treatments for visualization of the amplified nucleic acid. For example, the surface of the slide is treated so as to covalently bond 3-aminopropyl triethoxysilane, or the surface is coated with poly(lysine) or gelatin/chrome alum. The area of the slide with the specimen is then covered with PCR reagents. The slide and reagents are then cycled 10 to 40 times between temperatures typically between about 95° C. and 68° C., but sometimes as low as 37° C., spending at least a fraction of a minute or more at each of two or three selected temperatures during each cycle.
There are several important requirements that must be met during thermal cycling for in situ PCR to be successful. One is that evaporation of water from the PCR reagents must be prevented. No more than about 5% change from optimum PCR reagent concentrations can be tolerated without resulting in lower amplification yields or less specificity. Moreover, material which inhibits the PCR should be omitted from the process. In addition, bubbles of air or dissolved gas which are released by the reagents when they are heated should not disturb the access of the liquid reagent to the entire area to be processed. Furthermore, the conditions employed during the thermal cycling or subsequent processing to visualize the amplified nucleic acid should not disrupt tissue or cell morphology and should result in uniform and reproducible results.
Thus, in situ PCR requires a delicate balance between two opposite requirements of PCR in a cellular preparation: the cell and subcellular (e.g., nuclear) membranes must be permeabilized sufficiently to allow externally applied PCR reagents to reach the target nucleic acid, yet must remain sufficiently intact and nonporous to retard diffusion of amplified nucleic acid out of the cells or subcellular compartments where it is synthesized. In addition, the amplified nucleic acid must be sufficiently concentrated within its compartment to give a microscopically visible signal, yet remain sufficiently dilute that it does not reanneal between the denaturation and probe-annealing steps.
Nuovo et al. (U.S. Pat. No. 5,538,871) disclose that a commercially available thermal cycler, designed to accommodate multiple small plastic microcentrifuge tubes, can be modified to accommodate microscope slides. For example, it is disclosed that a single flat metal sample block can be machined to replace the top surface of a thermal cycler. It is also disclosed that the sample block can contain vertical slots in which the microscope slides are placed. However, Nuovo et al. do not disclose a device other than one having a metal sample block to perform PCR on microscope slides. Moreover, Nuovo et al. do not disclose a means to detect the temperature of the microscope slide during thermal cycling.
Lippman (U.S. Pat. No. 4,694,846) relates to a microscope slide system in which the slide is adapted for illuminating a sample in a depression on the upper surface of the slide. The sample has opaque particles suspended in a liquid, e.g., coal slurries. The Lippman patent discloses that the slide is heated by electrical resistive elements formed on the surface of the slide and a thermocouple may be attached to the slide so as to measure and control heat. The resistive elements and the thermocouple are each connected to an electrical source by wires. However, the Lippman patent does not mention a slide useful in a thermal cycling device, e.g., to amplify nucleic acids in a biological sample on the slide.
Thus, what is needed is an improved thermal cycling device for microscope slides.
SUMMARY OF THE INVENTION
The invention provi
Arik Mehmet
Bar-Cohen Avram
Blumenfeld Martin
Cibuzar Gregory T.
Schiller Peter
Lu Frank
Regents of the University of Minnesota
Westman Champlin & Kelly P.A.
Whisenant Ethan
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