Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-05-06
2001-04-24
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S005000, C435S006120
Reexamination Certificate
active
06221635
ABSTRACT:
BACKGROUND OF THE INVENTION
The ability to interrogate an unknown, small DNA sample with a relatively large number of questions and obtain a rapid yes
o complement of answers is highly desirable in many areas of molecular diagnostics. Developments in DNA and RNA amplification technology have enabled the design of very sensitive, high throughput methods (Saiki et al., 1985, Science 230:1350-1354). Polymerase chain reaction (PCR) amplification has been used to scan nucleic acid sequences for profiling or fingerprinting purposes using either predictable or arbitrary primers (Caetano-Anolles, 1996, Nature Biotech. 14:1668-1674). When predictable primers are used, the presence and distribution of repeated sequences in a genomic sample can be identified. Utilizing random primers, a multiplicity of anonymous sites in nucleic acid templates are scanned producing fingerprints or arbitrary collections of amplified products, giving rise to a specific profile of the genome being analyzed.
The possibility of interrogating an uncharacterized DNA sample for the presence of scarce copies of foreign DNA, or to determine whether it contains unique members of a large gene family or one of many possible genetic rearrangements is not yet satisfactorily resolved. Multiplex PCR (Chamberlain et al., 1988, Nucleic Acids Res. 16:11141-11156) could be utilized in theory, but if suffers from several limitations, the most serious being the undesirable interactions occurring with the increase in the number of primers in the multiplex PCR reaction. The alternative of carrying out hundreds of PCRs in parallel, each reaction containing primers specific for a defined gene sequence, is overly cumbersome, requiring highly specialized equipment and large amounts of reagents. The use of chips containing microarrays of diagnostic probes is only suitable if the gene of interest is sufficiently abundant in the sample and the procedure involves multiple steps (CDNA synthesis, PCR amplification, hybridization) that makes the procedure cumbersome. A more viable alternative would be to analyze a target DNA template using multiple sets of compartmentalized primer pairs anchored to a solid-phase, but a solid-phase PCR that can exponentially amplify target DNA has not yet been reduced to practice.
Methodologies of DNA template amplification have been developed in which either the nucleic acid template or the oligonucleotide primers are anchored to a solid-phase. In situ PCR wherein the nucleic acid template is immobilized on a nylon membrane, is an example (Skryabin et al., 1990, Nucleic Acids Res. 18:4289). Solid-phase anchored PCR is another example in which primers modified with amino links and coupled to solid-phase like agarose, acrylamide, magnetic beads (Jakobsen et al., 1990, Nucleic Acids Res. 18:36695) or latex beads (Kuribayashi-Ohta et al., 1993, Biochim. Biophys. Acta 1156:204-212) are used for a variety of applications. Such applications include the generation of immobilized cDNA suitable for automated sequencing of single stranded DNA (Hultman et al., 1989, Nucleic Acids Res. 17:4937-4946), the detection of products in automated clinical assays (Holmberg et al., 1992, Mol. Cell. Probes 6:201-208; Wahlberg et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:6569-6573), the generation of probes with high specific activity (Espelund et al., 1990, Nucleic Acids Res. 18:6157-6158), the construction of cDNA libraries (Tagle et al., 1993, Nature 361:751-753) and hybrid selection of RNA (Kuribayashi-Ohta et al., 1993, Biochim. Biophys. Acta 1156:204-212). Oligonucleotide probes, crosslinked to nylon, have been used for detection of specific PCR products (Saiki et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6230-6234) and anchored oligonucleotide primers have been used for detection of point mutations (Lockley et al., 1997, Nucleic Acids Res. 25:1313-1314). Other examples of solid-phase approaches are AMP-PCR (Wu et al., 1994, Nucleic Acids Res. 22:3257-3258), and RAMP (Zietkewicz et al., 1994, Genomics 20:176-183) using single anchored microsatellites primers.
Recently, a theoretical version of solid-phase PCR using bound forward and reverse primers and DNA template in solution has been proposed in a “bridge” PCR in a patent by Adams and Kron (U.S. Pat. No. 5,641,658). No data are presently available on whether solid-phase anchored primers are truly able to exponentially amplify a template or instead provide a linear amplification of the target template as the result of repeated primer extensions of the template by forward and reverse primer. In addition, there is no suggestion in the prior art that anchored primers and surface adsorbed DNA template on the same solid-phase could yield specific amplified product. Indeed, in light of the prior art teachings, such results are counterintuitive.
The present invention sets forth novel technology which provides a vast improvement to existing nucleic acid amplification methods and fills a long-felt need in the development of cost-effective and efficient methods of detecting low abundance genetic information in complex genomes.
SUMMARY OF THE INVENTION
The present invention relates to a method of detecting the presence or absence of a specific nucleic acid in a sample comprising DNA. The method comprises performing an amplification reaction, wherein a solid support on which a 5′ and a 3′ primer are irreversibly bound and the DNA is reversibly bound, is incubated under amplification conditions, and determining whether the specific nucleic acid is amplified, wherein when the specific nucleic acid is amplified, the specific nucleic acid is present in the sample and when the specific nucleic acid is not amplified, the specific nucleic acid is not present in the sample.
In one aspect, the solid support comprises a solid to which the DNA cannot be irreversibly bound upon adding the DNA to the solid support in the absence of any further reaction.
In another aspect, the solid support is selected from the group consisting of a nylon membrane, and a nitrocellulose membrane.
In a preferred embodiment, the solid support is a nylon membrane.
In another aspect, the primers are irreversibly bound to the solid support by UV-crosslinking the primers to the support.
In yet another aspect, the 5′ and 3′ primers further comprise a poly-thymidine sequence at their 5′ ends.
In another aspect, the poly-thymidine sequence comprises from about 15 to about 80 thymidine nucleotides.
In yet another aspect, the 5′ and 3′ primers comprise a sequence homologous to the sequence of the specific nucleic acid, the primers ranging in length from about 19 nucleotides to about 23 nucleotides.
In yet a further aspect, if the primers are in double stranded form, the thermal melting temperature of the double stranded form of the primers is in the range of about 55° C. to about 65° C.
In a preferred embodiment, the 5′ and 3′ primers are substantially lacking a GC rich region near the 3′ end of the 5′ and 3′ primers.
In one aspect, the amplification is performed by a reaction selected from the group consisting of a polymerase chain reaction, and a ligase reaction.
In another aspect, the determination of whether the specific nucleic acid is amplified is performed by incorporating a detectable label into the amplified nucleic acid during amplification and detecting the label.
In yet another aspect, the amplification conditions comprise about 2 mM MgCl
2
and about 50 mM KCl.
In yet a further aspect, the specific nucleic acid is selected from the group consisting of a nucleic acid comprising a mutation, a nucleic acid comprising a mutation in a cancer gene, and a nucleic acid comprising a sequence present in the genome of an infectious agent.
The invention also includes a method of determining the presence or absence of expression of a specific nucleic acid in a cell. The method comprises reverse transcribing RNA obtained from the cell to produce DNA, performing an amplification reaction, wherein a solid support on which a 5′ and a 3′ primer
Mukhopadhyay Sunil
Rovera Giovanni
Akin Gump Strauss Hauer & Feld L.L.P.
Einsmann Juliet C.
Fredman Jeffrey
The Wistar Institute
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