Analytical methods and materials for nucleic acid detection

Details Analytical methods and materials for nucleic acid detection Analytical methods and materials for nucleic acid detection

1999-11-22

2001-07-31

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007800, C435S091200, C435S091500, C436S173000, C436S501000, C536S026110, C536S027400, C536S028500

Reexamination Certificate

active

06268146

ABSTRACT:

TECHNICAL FIELD
The invention relates to nucleic acid detection. More specifically, the invention relates to the analytical methods for the detection of targeted, predetermined nucleic acid sequences in nucleic acid target/probe hybrids, and various applications of their detection.
BACKGROUND OF THE INVENTION
Methods to detect nucleic acids and to detect specific nucleic acids provide a foundation upon which the large and rapidly growing field of molecular biology is built. There is constant need for alternative methods and products. The reasons for selecting one method over another are varied, and include a desire to avoid radioactive materials, the lack of a license to use a technique, the cost or availability of reagents or equipment, the desire to minimize the time spent or the number of steps, the accuracy or sensitivity for a certain application, the ease of analysis, or the ability to automate the process.
The detection of nucleic acids or specific nucleic acids is often a portion of a process rather than an end in itself. There are many applications of the detection of nucleic acids in the art, and new applications are always being developed. The ability to detect and quantify nucleic acids is useful in detecting microorganisms, viruses and biological molecules, and thus affects many fields, including human and veterinary medicine, food processing and environmental testing. Additionally, the detection and/or quantification of specific biomolecules from biological samples (e.g. tissue, sputum, urine, blood, semen, saliva) has applications in forensic science, such as the identification and exclusion of criminal suspects and paternity testing as well as in genetics and medical diagnostics.
Some general methods to detect nucleic acids are not dependent upon a priori knowledge of the nucleic acid sequence. A luminescent nucleic acid detection method described in U.S. Pat. No. 4,735,897 is not sequence specific, indicates the presence of single-stranded RNA, such as mRNA. In the disclosed method, RNA is depolymerized using a polynucleotide phosphorylase (PNP) in the presence of phosphate or using a ribonuclease. That patent teaches that PNP stops depolymerizing when a double-stranded RNA segment is encountered, such as in single-stranded RNA with secondary structure, as is common in ribosomal RNA, transfer RNA, viral RNA, and the message portion of mRNA. PNP depolymerization of the polyadenylated tail of mRNA in the presence of inorganic phosphate forms ADP. Alternatively, depolymerization of RNA using a ribonuclease releases AMP. The released AMP is converted to ADP with myokinase, and ADP is converted into ATP by pyruvate kinase or creatine phosphokinase. Either the ATP or the byproduct from the organophosphate co-reactant (pyruvate or creatine) is detected as an indirect method of detecting mRNA. In U.S. Pat. No. 4,735,897, ATP is detected by a luminescence spectroscopic luciferase detection system. In the presence of ATP and oxygen, luciferase catalyzes the oxidation of luciferin, producing light that can then be quantified using a luminometer. Additional products of the reaction are AMP, pyrophosphate and oxyluciferin.
Hybridization methods to detect nucleic acids are dependent upon knowledge of the nucleic acid sequence. Many known nucleic acid detection techniques depend upon specific nucleic acid hybridization in which an oligonucleotide probe is hybridized or annealed to nucleic acid in the sample or on a blot, and the hybridized probes are detected.
Several hybridization methods to detect nucleic acids are discussed in the paragraphs that follow. These include PCR, Southern blots, and fluorescent hybridization with and without PCR. Several of hybridization methods are even useful for detecting specific nucleic acid sequences such as single nucleotide polymorphisms (SNPs), and distinguishing them from very similar sequences, and this is also discussed.
Polymerase chain reaction (PCR) and Southern blot-based hybridization methods for the detection of predetermined nucleic acid rely upon the use of hybridizing labeled primers or probes. Such probes have been labeled and detected using radioactivity; fluorescent spectroscopic methods using fluorescent dyes, acridinium esters and digoxygenin; and absorbance spectroscopic (often visible) methods using horseradish peroxidase, jack bean urease and alkaline phosphatase. PCR products made with unlabeled primers may be detected in other ways, such as electrophoretic gel separation followed by dye-based visualization.
There are hybridization assays to detect nucleic acid that involve fluorescence spectroscopic techniques utilizing energy transfer effects between fluorophores (FRET). U.S. Pat. No. 5,691,146 describes the use of fluorescent hybridization probes that are fluorescence-quenched unless they are hybridized to the target nucleic acid sequence. U.S. Pat. No. 5,723,591 describes fluorescent hybridization probes that are fluorescence-quenched until hybridized to the target nucleic acid sequence, or until the probe is digested. Such techniques provide information about hybridization, and are of varying degrees of usefulness for the determination of single base variances in sequences. Some fluorescence techniques involve digestion of the probe in a nucleic acid hybrid in a 5′ to 3′ direction to release a fluorophore providing a signal from proximity with a fluorescence quencher, thereby increasing the signal, for example, U.S. Pat. Nos. 5,691,146 and 5,876,930.
The fluorescence spectroscopic techniques for detection of nucleic acid hybrids have been applied to real-time (or kinetic) PCR and single nucleotide polymorphism (SNP) detection. Many of these systems are platform based and require specialized equipment, complicated primer design and expensive supporting materials for SNP detection. SNP detection using real-time PCR amplification relies on the ability to detect amplified segments of nucleic acid as they are made during the amplification reaction. Three basic real-time SNP detection methodologies exist: (i) increased fluorescence of double-stranded DNA-specific dye binding, (ii) decreased quenching of fluorescence during amplification (e.g. Taqman®), and (iii) increased fluorescence energy transfer during amplification (C. Wittwer et al.,
Biotechniques,
22:130-138 (1997)). All of these techniques are non-gel based and each strategy is briefly discussed below.
A variety of dyes are known to exhibit increased fluorescence in response to binding double stranded DNA. Production of wild type or mutation containing-PCR products are continuously monitored by the increased fluorescence of dyes such as ethidium bromide or syber green as they bind to the accumulating duplex PCR product. Note that dye binding is not selective for the sequence of the PCR product, and elevated levels of non-specific background can give rise to false signals with this technique.
Some technologies for real time detection of PCR products are based on detecting nucleic acid hybrids using fluorescence resonance energy transfer (FRET; mentioned above). These technologies either indirectly measure the amplification reaction through the use of a separate, labeled probe that hybridizes with but is not incorporated into the amplification product (U.S. Pat. Nos. 5,348,853; 5,119,801; 5,312,728; 5,962,233; 5,945,283; 5,876,930; 5,723,591; and 5,691,146) or directly detect amplification products through the use of a label directly incorporated in the amplification primer(s) (U.S. Pat. No. 5,866,336).
One such FRET-based technology for real time PCR product detection is known generally as 5′ nuclease PCR assay (TaqMan® assay). In this assay the decrease in fluorescence quenching resulting from the cleavage of dually-labeled probes that hybridize downstream of amplification primers is monitored in an amplification reaction. A polymerase extends the growing nucleic acid chain from the amplification primers, and degrades hybridized dually-labeled probes from their 5′-termini using the 5′ to 3′ exonuclease activity of therm

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