Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-18
2004-09-07
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S094000, C536S025400
Reexamination Certificate
active
06787310
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to compositions and methods for isolating one strand of double-stranded nucleic acids.
2. BACKGROUND
Many techniques in the field of molecular biology involve hybridizing a target nucleic acid to a support-bound or solution-phase single-stranded oligonucleotide probe for analyzing, capturing, isolating and/or detecting the target nucleic acid. Such techniques range from Sanger-type DNA sequencing (see, e.g., Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467; Ansorge et al., 1987, Nucl. Acids Res. 15:4593-4602; Smith et al., 1985, Nucl. Acids Res. 13:2399-2412; Smith, et al., 1986, Nature 321:674-679; Prober et al., 1987, Science 238:336-341), where a labeled or unlabeled primer is annealed to one strand of a target and enzymatically extended in the presence of 2′,3′-dideoxyribonucleotide terminators (see also, Carrilho, 2000, Electrophoresis 21:55-65 and Kheterpal & Mathies, 1999, Anal. Chem. 71:31A—37A) to array-based gene expression, genotyping, gene mapping and nucleic acid sequencing assays (see, e.g., U.S. Pat. Nos. 5,202,231, 5,695,940 and 5,525,464, WO 95/09248, Khrapko et al., 1991, DNA Sequence 1:375-388; Southern et al., 1992, Genomics 13:1008-1017; Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; for reviews of the various array-based assays commonly employed in the art see Thompson & Furtado, 1999, Analyst 124:1133-1136; Rockett & Dix, 2000, Xenobiotica 30:155-177; Granjeaud et al., 1999, Bioessays 21:781-790; Lipscutz et al., 1999, Nat. Genet. 21(1 Suppl.):20-24; DeRisi & Iyer, 1999, Curr. Opin. Oncol. 11:76-79; Blanchard, 1998, Genet. Eng. 20:111-123; Case-Green et al., 1998, Curr. Opin. Chem. Biol. 2:404-410; Johnston, 1998, Curr. Biol. 8:R171-174; de Saizieu et al., 1998, Nat. Biotechnol. 16:45-48; and Marshall & Hodgson, 1998, Nat. Biotechnol. 16:27-31).
When the target nucleic acid is single-stranded, hybridization to the oligonucleotide probe occurs relatively easily. But when the target nucleic acid is double-stranded, reassociation of the two target strands strongly competes with, and usually out-competes, hybridization between the target and oligonucleotide probe, especially under the high-salt stringent conditions commonly employed in these assays.
Unfortunately, target nucleic acids are rarely available in single-stranded form. Indeed, most target nucleic acids are double-stranded. For example, genomic DNA, genomic fragments and cDNA are double-stranded. Moreover, one of the most commonly-used amplification techniques for generating analyzable quantities of a specific nucleic acid of interest, the polymerase chain reaction (“PCR” see, e.g., U.S. Pat. No. 4,683,202; Sambrook et al., 2d ed. 1989, M
OLECULAR
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: A L
ABORATORY
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, Cold Springs Harbor, N.Y.) generates double-stranded amplicons, or target nucleic acids.
Current methods for generating single-stranded target nucleic acids include exonuclease digestion of one strand of the double-stranded target (see, e.g., Hannon et al., 1993, Anal. Biochem. 212:421-427), asymmetric PCR amplification (see, e.g., Stürzl and Roth, 1990, Anal. Biochem. 185:164-169), generating single-stranded RNA targets by in vitro transcription of double-stranded PCR amplicons having a T7 or T3 RNA polymerase promoter (see, e.g., Yang and Melera, 1992, BioTechniques 13:922-927) and cloning with Ml 3 (see, e.g., Sambrook, et al., supra). Each of these methods has significant drawbacks that either limit its general applicability and/or significantly increase the time and expense of the assay.
Accordingly, there remains a need in the art for simple methods of generating and isolating one strand of a double-stranded target nucleic acid for assays involving hybridization with a single-stranded oligonucleotide probe, such as Sanger-type sequencing reactions, and array-based mapping, genotyping, expression and sequencing applications.
3. SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method for capturing or isolating one strand of a double-stranded target nucleic acid. The method is based, in part, on the observations that the thermodynamic stabilities and/or kinetics of formation of nucleic acid duplexes under varying conditions of ionic strength depend upon the nature of the internucleoside linkages comprising the strands of the duplex. For example, as evidenced by the occurrence of double-stranded DNAs in nature and the ability of DNAs and RNAs to form homo and hybrid duplexes under physiological conditions, duplexes formed between single-stranded nucleic acids having like-charged internucleoside linkages, such as DNAs and RNAs which have negatively charged sugar phosphodiester interlinkages, are relatively stable under conditions of physiological ionic strength (approximately 100 mM NaCl), temperature (approximately 37° C.) and pH (approximately pH 7.2). However, under conditions of low ionic strength (approximately 10 mM NaCl or lower), such DNA/DNA, RNA/RNA and DNA/RNA duplexes tend to dissociate (see, e.g., Egholm et al., 1993, Nature 365:566-568). While not intending to be bound by any particular theory, it is believed that the observed dissociation at conditions of low ionic strength is due to interstrand electrostatic repulsion caused by the negatively charged sugar phosphodiester nucleobase interlinkages.
On the contrary, heteroduplexes in which one strand of the duplex is a conventional DNA or RNA and the other strand is a nucleic acid analog that has a net uncharged or net positively charged backbone (e.g., a PNA) are quite stable at conditions of low ionic strength (see, e.g., id.). In fact, at NaCl concentrations between about 0 and 10 mM, and even as high as 500 mM, such heteroduplexes are significantly more stable than their corresponding DNA/DNA, RNA/RNA or DNA/RNA duplexes (id).
Moreover, the formation of these heteroduplexes is favored kinetically over the formation of the corresponding DNA/DNA, DNA/RNA and RNA/RNA duplexes under the same conditions of low ionic strength. These observed differences in kinetics are independent of the length of the heteroduplex. For example, while a relatively long DNA/DNA target duplex (e.g., ≧100 bp) is typically more thermodynamically stable than a short PNA/DNA heteroduplex (e.g., ≦20 bp) at virtually any ionic strength, if the DNA/DNA target duplex is dissociated and contacted with even a short complementary PNA under conditions of low ionic strength (e.g., less than 10 mM NaCl), the formation of the PNA/DNA heteroduplex will be kinetically favored over the reannealing or reassociation of the DNA target strands. Only as the system is brought to equilibrium will the PNA be displaced by the complementary DNA target strand.
The methods of the invention capitalize on these observed kinetic and thermodynamic stability differences to easily and efficiently isolate one strand of a double-stranded target nucleic acid. Generally, the method involves contacting a double-stranded target nucleic acid with a single-stranded competitor oligonucleotide (“competitor oligo”) that is capable of hybridizing to one strand of the double-stranded target. The competitor oligo is a nucleic acid analog which comprises a combination of negatively charged (e.g., a native sugar phosphodiester), positively charged (e.g., a sugar glycosyl or positive amide) and/or uncharged (e.g., neutral amide or morpholino-phosphoramidate) nucleobase interlinkages such that the competitor oligo has a net positive charge or a net neutral charge at the desired pH and temperature of use (typically pH 6-9 and 20-40° C.). Preferably, the competitor oligo is wholly composed of uncharged nucleobase interlinkages, and optionally includes 3-4 positive interlinkages. The nucleobase sequence of the competitor oligo is at least partially complementary to a portion of one strand of the double-stranded target such that it can hybridize to its complementary target strand.
The target nucleic acid is contacted with the competitor oligo under conditions in which the target strands tend to dissociate from
Chiesa Claudia
Egholm Michael
Schroth Gary P.
Applera Corporation
Bortner Scott R.
Finn Andrew K.
Horlick Kenneth R.
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