Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1997-11-12
2004-11-09
Warden, Jill (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S307000, C250S458100
Reexamination Certificate
active
06814934
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention provides improved methods for nucleic acid detection. The novel methods for simultaneous nucleic acid amplification and detection enhance the speed and accuracy of prior detection methods and eliminate the need for sample processing following amplification. In a preferred embodiment, the method provides a modification of the polymerase chain reaction and utilizes agents whose fluorescence is enhanced upon binding double-stranded DNA. The methods provided herein have numerous applications, particularly in the fields of molecular biology, medical diagnostics and forensic sciences.
Description of Related Art
The disclosed nucleic acid detection methods offer the advantages of speed and simplicity over prior methods for detecting amplified nucleic acids. Nucleic acid detection techniques in general are particularly useful in medical diagnostic assays. For example, Falkow et al., U.S. Pat. No. 4,358,535 disclose a method for detecting pathogens by spotting a sample (e.g., blood, cells, saliva, etc.) on a filter, lysing the cells and fixing the DNA through chemical denaturation and heating. Then, labeled DNA probes are added and allowed to hybridize with the fixed sample DNA. Hybridization indicates the presence of the pathogen's DNA.
Nucleic acid detection using oligonucleotide probes has become a standard method for specific target detection. Numerous modifications of the method have been described, including culturing the target cells or organisms in situ on the filter, increasing the amount of target nucleic acid available for detection. Generally, these methods require that the DNA sample is noncovalently bound onto a solid support such as nitrocellulose or nylon and then hybridized to a labeled target-specific probe.
The sensitivity and specificity of nucleic acid detection methods was greatly improved by the invention of the polymerase chain reaction (PCR). PCR is a process for amplifying nucleic acids and involves the use of two oligonucleotide primers, an agent for polymerization, a target nucleic acid template, and successive cycles of denaturation of nucleic acid and annealing and extension of the primers to produce a large number of copies of a particular nucleic acid segment. With this method, segments of single copy genomic DNA can be amplified more than 10 million fold with very high specificity and fidelity. PCR methods are disclosed in U.S. Pat. No. 4,683,202, which is incorporated herein by reference.
Methods for detecting PCR products are particularly described in U.S. Pat. No. 4,683,195, which is incorporated herein by reference. Those methods require an oligonucleotide probe capable of hybridizing with the amplified target nucleic acid. European Patent Publication No. 237,362, which is incorporated herein by reference, also describes a PCR-based detection method termed “reverse dot blot”, in which the probe, instead of the amplified DNA, is fixed to the membrane. According to the method, the target, rather than the probe, is labeled for hybridization. These methods require separate steps of amplification, capture, and detection and generally require several hours to complete. In the reverse dot-blot method, storage-stable target-specific reagents are preferred.
Alterative methods for detecting amplified nucleic acids are described in copending U.S. Ser. No. 076,394, filed Jul. 22, 1987, which is incorporated herein by reference. U.S. Ser. No. 076,394 describes PCR-based methods for simultaneous amplification and labeling of a target nucleic acid. The methods require that at least one amplification primer is labeled. The amplification primer can be labeled with, for example, a radioisotope for direct detection of the amplified product or labeled with a reagent suitable for capturing the product onto a solid support for subsequent detection.
Other means of detection include the use of fragment length polymorphism hybridization, allele-specific oligonucleotide (ASO) probes (Saiki et al., 1986,
Nature
324:163), or direct sequencing via the dideoxy method using amplified DNA rather than cloned DNA. The fragment length polymorphism method detects insertions and deletions between PCR primers resulting in PCR products of different lengths, detectable by sizing. ASO methods are useful for detecting allelic sequence variations. In an example of ASO hybridization, the amplified DNA is fixed to a nylon filter (by, for example, UV irradiation) in a series of “dot blots,” then allowed to hybridize with an oligonucleotide probe under stringent conditions. This method is also described in copending U.S. Ser. No. 347,495, filed May 4, 1989, which is incorporated herein by reference. The probe may be labeled with, for example, horseradish peroxidase (HRP) and detected by the presence of a blue precipitate following treatment with suitable oxidation reagents.
Copending U.S. Ser. No. 563,758, filed Aug. 6, 1990 (now U.S. Pat. No. 5,210,015), and incorporated herein by reference, describes an alterative assay method for detecting amplified nucleic acids. The process employs the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed, labeled oligonucleotides from hybridized duplexes and release labeled oligonucleotide fragments for detection. The method is suitable for detecting PCR products and requires a primer pair and a labeled oligonucleotide probe having a blocked 3′—OH terminus to prevent extension by the polymerase.
Due to the enormous amplification possible with the PCR process, small levels of DNA carryover from samples with high DNA levels, positive control templates, or from previous amplifications, can result in PCR product even in the absence of purposefully added template DNA. Higuchi and Kwok, (1989,
Nature
339:237-238 and Kwok) and Orrego, (in Innis et al., 1990
, PCR Protocols: A Guide to Methods and Applications
, Academic Press, Inc., San Diego, Calif.), describe particular methods and precautions for practicing PCR with a minimum of cross contamination. U. S. Ser. No. 609,157, filed Nov. 2, 1990, describes improved methods for reducing the effects of cross contamination by the introduction of unconventional nucleotide bases. These references are incorporated herein by reference. Because the possibility of introducing contaminating DNA to a sample will be increased as the amount of handling steps required for sample preparation, processing, and analysis is increased, it would be preferable to minimize sample handling, particularly after the amplification reaction is complete.
A number of agents have been described for labeling nucleic acids, whether probe or target, for facilitating detection of target nucleic acid. Suitable labels may provide signals detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism or enzymatic activity and include, for example, fluorophores, chromophores, radioactive isotopes (particularly
32
P and
125
I) electron-dense reagents, enzymes, and ligands having specific binding partners.
Labeling is achieved by a number of means, such as chemical modification of a primer or probe to incorporate a label or the use of polymerizing agents to incorporate a modified nucleoside triphosphate into an extension product. Intercalating agents non-covalently bind the stacked bases of nucleic acids and as a result the fluorescence of the agent either increases or shifts to a different wavelength. For example, U.S. Pat. No. 4,582,789 describes several intercalating moieties including psoralens. Copending U.S. Ser. No. 076,394 describes methods for amplifying and detecting nucleic acids using psoralen labeled primers. Both the '789 patent and the '394 application are incorporated herein by reference.
Fluorescent dyes are suitable for detecting nucleic acids. For example, ethidium bromide is an intercalating agent that displays increased fluorescence when bound to double-stranded DNA rather than when in free solution (Sharp et al., 1973,
Biochemistry
12:3055). Ethidium bromide can be
LandOfFree
Instrument for monitoring nucleic acid amplification does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Instrument for monitoring nucleic acid amplification, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Instrument for monitoring nucleic acid amplification will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3354474