WO1999001570A2

WO1999001570A2 - Methods for accelerating hybridization of nucleic acid molecules

Info

Publication number: WO1999001570A2
Application number: PCT/CA1998/000631
Authority: WO
Inventors: Robert N. Bryan; Lynn P. Cloney; Barbara A. Farnworth; A. Johanna Marostenmaki
Original assignee: Id Biomedical Corporation
Priority date: 1997-07-03
Filing date: 1998-07-03
Publication date: 1999-01-14
Also published as: AU8327298A; WO1999001570A3

Abstract

A method of increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule is provided, comprising the step of mixing a first nucleic acid molecule, a second nucleic acid molecule, and polyamine and/or RNase H under conditions and for a time sufficient to permit hybridization of said first nucleic acid molecule to the second nucleic acid molecule.

Description

METHODS FOR ACCELERATING HYBRIDIZATION OF NUCLEIC ACID MOLECULES

TECHNICAL FIELD

The present invention relates generally to methods for improving the rate of hybridization of complementary nucleic acid molecules, and in particular towards methods for accelerating nucleic acid hybridization reactions utilizing ribonuclease H and/ or polyamines.

BACKGROUND OF THE INVENTION

There are a number of techniques available in the diagnostic field for detecting pathogenic organisms or genetic disease within a biological sample, including for example, biochemical tests, immunological tests and cytological tests. The majority of these techniques, however, have drawbacks related to time, quantity of sample required and lack of specificity or sensitivity of detection. To address these issues, techniques which involve direct detection of nucleic acids through nucleic acid hybridization have been developed.

Briefly, technologies involving nucleic acid hybridization are useful for detecting, amplifying, or isolating a selected nucleic acid sequence. Nucleic acid hybridization occurs when a single stranded nucleic acid molecule (e.g., a "probe") forms a duplex with a complementary sequence or substantially complementary sequence present in another nucleic acid molecule (e.g., a "target"). Such duplex formation can take place in solution, when one nucleic acid molecule is immobilized to a solid support, or in situ. For purposes of detecting, duplex nucleic acids are separated from single-stranded nucleic acids, and presence of the duplex is detected (e.g., by detecting the presence of a labeled probe). Nucleic acid hybridization reactions are utilized in a wide variety of amplification reactions, including for example, Polymerase Chain Reaction ("PCR") (Erlich (ed.) PCR Technology, Stockton Press, 1989; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), ligase chain reaction (U.S. Pat. No. EP-A-320 308; WO 90/01069), strand displacement amplification (Walker et al., Nucleic Acids Res. 20:1691-1696, 1992), branched-chain DNA signal amplification (bDNA, Urdea, Clin. Chem. 39:125- 726, 1993), Q-beta-replicase (Lizardi et al., Biotechnology (5:1197-1202, 1988), transcription-based amplification systems such as Transcriptional Amplification System (TAS, Kwoh et al, Proc. Natl. Acad. Sci USA 56:1173-1177, 1989), Self Sustained Sequence Replication (3SR, Guatelli et al, Proc. Natl. Acad. Sci USA §7:1874-1978. 1990), Nucleic Acid Sequence Based Amplification (NASBA, U.S. Pat. No. 5,409,818), Transcription-Mediated Amplification (TMA, U.S. Pat. No. 5,399,491), and 5' nuclease based amplification assays (e.g., Lyamichev et al., Science 260:778-783, 1993).

One difficulty with present hybridization reaction methods, however, is that in dilute solutions of nucleic acids, the half time (t_1/2) of hybridization is extremely long. Several means have been suggested for improving hybridization rates, including increasing salt concentration (Wetmur & Davidson, J. Mol. Biol. 31:349-310, 1968; Kohne, U.S. Pat. No. 5,132,207) and increasing detergent concentrations (Kohne, supra). In addition, use of inert polymers, such as dextran sulfate or polyethylene glycol, or phenol emulsions (see Wetmur, Crit. Rev. Biochem. Mol. Biol, 26:221, 1991; and Sikorav et al., J. Mol. Biol. 222:1085-1108, 1991) has also been suggested.

Other methods for increasing hybridization rates include use of a large excess of one complementary strand to drive the reaction (Anderson and Young, in Hanes and Higgins (Eds.), Nucleic Acid Hybridization a practical approach, IRL Press, Oxford, 1987, pp. 73-111; Wetmur, Crit. Rev. Biochem. Mol. Biol 26:221, 1991). Excess probe nucleic acid molecules are often used for detecting target nucleic acid molecules which may be in solution or immobilized (Keller, in Keller and Manak, DNA probes, Second edition, Stockton Press, New York, 1993, pp. 1-25). One disadvantage of this approach, however, is that when the excess probe is labeled, even though the reaction rate may be faster, there may be a concomitant increase in background.

Although the above methods can increase hybridization rates for certain types of nucleic acid reactions, there is a need for a method that applies to both natural and non-natural nucleic acid molecules with increased specificity, decreased background and reduction in assay times. The present invention provides methods for accelerating hybridization rates of nucleic acid molecules, and further, provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides novel compositions and methods for increasing hybridization rates of nucleic acid molecules. Within one aspect of the present invention, methods are provided for increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule, comprising the step of mixing a first nucleic acid molecule, a second nucleic acid molecule and a polyamine under conditions and for a time sufficient to permit hybridization of said first nucleic acid molecule to said second nucleic acid molecule. Within another aspect of the present invention, methods are provided for increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule, comprising the step of mixing a first nucleic acid molecule, a second nucleic acid molecule and a polyamine wherein the polyamine (spermine or spermidine) is present at concentrations of less than lOmM or 20mM (e.g., 0.01 mM to 8 mM for spermine, and less than 5mM or lOmM for spermidine) (without salts such as NaCl, KC1, MgCl₂, or, with only minimal salts), under conditions and for a time sufficient to permit hybridization of said first nucleic acid molecule to said second nucleic acid molecule.

Within one embodiment of the invention the polyamine can be spermine or spermidine. Within other embodiments, the nucleic acid molecule (either the first or second, or any portion of these) may be composed of DNA, RNA, or at least one phosphonate, diphosphonate, phosphorotriester, phosphoramidite, 2'-O alkyl or aryl ribonucleotide.

Within another embodiment, the reaction mixture can further comprise minimal or no standard hybridizations salts, such as NaCl, KC1 or MgCl₂.

Within further embodiments, the first nucleic acid molecule is shorter than the second nucleic acid molecule, and the method may further comprise the step of extending the first nucleic acid molecule along the second nucleic acid molecule. Within other embodiments the first or second nucleic acid molecules may be in solution, or, bound to a solid support (where a further step of washing may be required).

Within other related embodiments, such methods may further comprise the step of passing the mixture through a polyacrylamide gel. Within yet other embodiments, the first or second nucleic acid molecule can be labeled with, for example, a member of specific binding pair, chemiluminescence, fluorescent molecules, coenzymes, enzyme substrates and radioactive groups.

Within other, further embodiments of the invention, the mixture can further comprise RNase H (e.g., a thermostable RNase H such as T. thermophilus RNase H), or a non-thermostable RNase H (such as E. coli RNase H). Within further embodiments, the mixture further comprises Na⁺ ion equivalent to 0 mM to 100 mM sodium phosphate, and/or a chelator such as EDTA or EGTA.

Within other aspects of the invention, methods are provided for increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule, comprising the step of mixing a first nucleic acid molecule, a second nucleic acid molecule and RNase H under conditions and for a time sufficient to permit hybridization of said first nucleic acid molecule to said second nucleic acid molecule, with the proviso that said first and second nucleic acid molecule does not contain scissile linkage, and, with the further proviso that if said first nucleic acid is DNA, that the second nucleic acid molecule is not RNA, or if the first nucleic acid is RNA, the second nucleic acid molecule is not DNA.

Within various embodiments of the invention, RNase H can be thermostable (e.g., T. thermophilus RNase H), or non-thermostable (such as E. coli

RNase H). Within further embodiments, the mixture further comprises Na⁺ ion equivalent to 0 mM to 100 mM sodium phosphate, and/or a chelator such as EDTA or

EGTA.

Within one embodiment, the reaction mixture can further comprise a polyamine such as spermine or spermidine.

Within another embodiment, the reaction mixture can further comprise minimal or no standard hybridizations salts such as NaCl, KC1 or MgCl₂.

Within other embodiments, the nucleic acid molecule (either the first or second, or any portion of these) may be composed of DNA, RNA, or at least one phosphonate, diphosphonate, phosphorotriester, phosphoramidite, 2'-O alkyl or aryl ribonucleotide.

These and other aspects of the present invention will become evident upon reference to the following detailed description. In addition, various references are set forth herein which describe in more detail certain procedures or compositions, and are therefore incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

Prior to setting forth the invention, it may be helpful to an understanding thereof to first set forth definitions of certain terms that will be used hereinafter. "Nucleic acid molecule" refers to a polymeric nucleotide or polynucleotide, which can have a natural or synthetic origin. Representative examples of nucleic acid molecules include DNA (ds- or ss-DNA), RNA, DNA-RNA hybrids, or nucleic acid molecules which are composed of or contain a nucleic acid analogue (e.g. , α-enantiomeric forms of naturally-occurring nucleotides). Furthermore, nucleo tides may be modified in their sugar moities, or in the pyrimidine or purine base moities. Examples of modification to sugar moities include modification or replacement of, for example, one or more hydroxyl groups with another group. Modifications to base moities include alkyl or acylated pyrimidines and purines. In addition, nucleic acid monomers can be linked by phosphodiester bonds, or analogs of such linkages (e.g., phosphorothioate, phosphorodithioate, phosphoramidite, and the like.

"Isolated nucleic acid molecule" refers to a nucleic acid molecule thatis not integrated into the genomic DNA of an organism. Isolated nucleic acid molecules include, for example, probes and other synthetically or recombinantly generated nucleic acid molecules. "Probe" refers to a synthetic oligonucleotide, usually single stranded, which is selected in view of known sequence to be complementary or substantially complementary to the target nucleic acid molecule to be detected. The probe is usually comprised of a sequence of at least 6 nucleotides, preferably 8, 10, 12, 14, 15, 16, 18 or 20 nucleotides, and can be up to 100 nucleotides or more. Within certain embodiments, the probe may be labeled.

"Primer" refers to a short synthetic oligonucleotide, usually a single stranded first nucleic acid molecule, that is utilized in a chain extension reaction based on the second nucleic acid molecule as the template.

"Scissile linkage" refers to a nucleic acid molecule which is capable of being cleaved or disrupted without cleaving or disrupting any nucleic acid sequence of the molecule itself or of the target nucleic acid sequence. Scissile linkages include any connecting chemical structure which joins two nucleic acid sequences and which is capable of being selectively cleaved without cleavage of the nucleic acid sequences to which it is joined. The scissile linkage may be a single bond or a multiple unit sequence. An example of such a chemical structure is an RNA sequence. Other chemical structures suitable as a scissile linkage are a DNA sequence, an amino acid sequence, an abasic nucleotide sequence or an abasic nucleotide, or any carbohydrate polymer, i.e., cellulose or starch. When the scissile linkage is a nucleic acid sequence, it differs from the nucleic acid sequences of NA, and NA₂ (described below). "Ribonuclease H" ("RNase H"): refers to an enzyme capable of specifically cleaving the RNA strand in RNA:DNA hybrid duplex (see generally Crouch & Dirksen in Nucleases, Linn & Roberts (Eds.), pp. 211-241, Cold Spring Harbour Laboratory Press, Plainview, NY., 1982).

The present invention provides means of rapidly increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule, utilizing additives such as polyamines, RNase H or a combination of both. Hence, as noted above, within one aspect of the present invention, methods are provided for increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule, comprising the step of mixing a first nucleic acid molecule, a second nucleic acid molecule and RNase H or a polyamine under conditions and for a time sufficient to permit hybridization of said first nucleic acid molecule to said second nucleic acid molecule.

A. Nucleic Acid Molecules

A wide variety of nucleic acid molecules may function as either the first or second nucleic acid molecule as described herein. Representative examples of nucleic acid molecules useful in this invention include both natural occurring and recombinant or synthetic nucleic acid molecules. More specifically, nucleic acid molecules may be purified or unpurified, and may be obtained from natural sources such as genomic or mitochondrial nucleic acids.

Moreover, suitable nucleic acid molecules can be obtained from viruses, prokaryotes (e.g., bacterial cells) or eukaryotes (e.g., yeast or parasites), as well as a wide variety of mammalian cells. Methods for extracting and purifying nucleic acids from various organisms should be evident in the disclosure provided herein. As an illustration, Example 3 is a method for purifying bacterial genomic DNA from Staphylococcus aureus. Nucleic acid molecules can also be constructed utilizing a wide variety of techniques (see, e.g., Sambrook et al, Molecular Cloning, Cold Spring Harbor Press, 1980). For example, within one embodiment nucleic acid molecules can be constructed on a solid support medium (such as silica gel or controlled pore glass) using either a hydrolysable linkage or a permanent (non-hydrolysable) linkage. Nucleic acid molecules can also be constructed as generally described by Matteucci and Caruthers, J. Am. Chem. Soc. 103:3\%5, 1981; Beaucage and Caruthers, Tetrahedron Lett. 22:1859, 1981; Alvarado-Urbina et al., "Automated Synthesis of Gene Fragments," Science 214:210-21 '4, 1981; Van Boom and Wreesman, "Chemical Synthesis of Small Oligoribonucleotides in Solution," In Oligonucleotide Synthesis - A Practical Approach, IRL Press, Gait (ed.), pp. 153-183, 1984; Agrawal (ed.) Protocols For Oligonucleotides And Analogs, Synthesis; Synthesis and Properties, Methods in Molecular Biology Volume 20, Humana Press Inc., 1993; Agrawal (ed.) Protocols For Oligonucleotide Conjugate, Synthesis And Analytical Techniques, Methods in Molecular Biology Volume 26, Humana Press Inc., 1994. Particularly preferred probes for use within the present invention include cleavable oligonucleotide probes (e.g., probes with a scissile linkage, see U.S. Patent Nos. 4,876,187; 5,011,769 and 5,403,711), standard nucleic acid molecules, analogs or peptide nucleic acids.

Briefly, oligonucleotide synthesis can be accomplished in cycles wherein each cycle extends the oligonucleotide by one nucleotide. Each cycle consists of four steps: (1) deprotecting the 5'-terminus of the nucleotide or oligonucleotide on the solid support; (2) coupling the next nucleoside phosphoroamidite to the solid phase immobilized nucleotide; (3) capping the small percentage of the 5'-OH groups of the immobilized nucleotides which did not couple to the added phosphoramidite; and (4) oxidizing the oligonucleotide linkage to a phosphotriester linkage.

B. Ribonuclease H

Ribonuclease H (RNase H) occurs in organisms ranging from prokaryotes to eukaryotes (reviewed by Crouch & Dirksen in Nucleases, Linn & Roberts (Eds.), pp. 211-241, Cold Spring Harbour Laboratory Press, Plainview, NY., 1982). RNase H can be obtained commercially, or prepared according to known techniques. In particular, RNase H can be isolated and purified from thermophilic and non-thermophilic organisms (see for example Kanaya et al., J. Bio. Chem. 258:1216- 1281, 1983; Kanaya & Itaya, J. Biol. Chem. 267:10184-10192, 1992). RNase H useful for this invention can be obtained from thermophilic bacteria such as Thermus thermophilus or alternatively, the RNase H gene can be cloned and expressed in E. coli by the method of Kanaya & Itaya, supra. Recombinant technologies can also be used for thermostablizing RNase H variants from non-thermostable organisms (Ishikiwa et al., Protein Eng. 6:85-91, 1993). Non-thermostable RNase H useful in this invention can be isolated and purified from E.coli by the method of Kanaya et al., supra. T. thermophilus and E.coli RNase H are also available commercially. T. thermophilus RNase H has greater residual activity at 65°C (Itaya & Kondo, Nucl. Acids Res. 76:4443-4449, 1991) and has 34°C higher thermal unfolding temperature than the E. coli enzyme (Ishikawa et al., J. Mol. Biol. 230:529542, 1993). RNase H requires divalent cations for its catalytic activity (Crouch and Dirksen, supra). C. Polyamines

Polyamines are naturally occurring, polybasic compounds which have higher affinity for acidic constituents (negatively charged) compared to monoamines or cations such as Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺ (see generally Tabor & Tabor, Ann. Rev. Biochem. 53:749-790, 1984). Spermidine and spermine are polyamines with three and four positive charges, respectively.

As discussed in more detail below, one aspect of the present invention is based on the unexpected discovery that polyamines by themselves also have the capacity to rapidly accelerate nucleic acid hybridization reactions. This ability applies to different types, amounts and sizes of nucleic acid molecules ranging from short synthetic nucleic acid molecules to long nucleic acid molecules. Polyamines can be utilized within the context of the present invention without the need for monovalent or divalent cations.

D. Hybridization Methods As noted above, the present invention provides methods for increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule, comprising the step of mixing a first nucleic acid molecule, a second nucleic acid molecule and RNase H and/or polyamine under conditions and for a time sufficient to permit hybridization of said first nucleic acid to said second nucleic acid molecule. The rate constant of hybridization of nucleic acid molecules can be readily obtained by a number of methods known in the art including: hydroxylapatite binding, SI nuclease assay, optical hyperchromicity or gel electrophoresis. One representative method for measuring rate constant is described in more detail below in Example 4.

General hybridization reactions and conditions are well known in the art, see generally Hanes and Higgins (Eds.), Nucleic Acid Hybridization a practical approach, IRL Press, Oxford, 1987; Keller and Manak, DNA probes, Second edition, Stockton Press, New York, 1993; Gilmartin, Nucleic acid hybridization essential data, John Wiley and Sons, West Sussex, 1996. The kinetics of hybridization reaction and methods for measuring them are also well known in the art (see generally Kohne and Britten, in Cantoni and Davies (Eds), Procedures in Nucleic Acid Research, Volume 2, Harper and Row, New York, pp. 500-512, 1971; Britten and Davidson, in Hanes and Higgins (Eds.) supra, pp. 3-15; Wetmur, Crit. Rev. Biochem. Mol. Biol, 26:221, 1991). Hybridization reactions follow second order kinetics if the first and second nucleic acid molecules are present in equimolar concentrations and pseudo-first order if one of the nucleic acid molecule is in excess and drives the hybridization reaction. Increasing the concentration of one nucleic acid molecule ('driver') in hybridization reaction results in faster hybridization reactions.

RNase H and/or polyamine may be utilized in a wide array of hybridization reactions. For example, in one embodiment both the first and second nucleic acid molecules are from a natural source of genomic or chromosomal dsDNA that is denatured. In other embodiments, the two nucleic acid molecules are synthetic in origin, or the first nucleic acid is synthetic in origin (e.g., is a probe or a primer), and the second molecule is from a natural source (e.g., genomic DNA). In further embodiments, the first nucleic acid is DNA and the second nucleic acid is RNA, or the first nucleic acid molecule is a chimeric nucleic acid molecule and the second nucleic acid is natural or synthetic in origin. Within certain embodiments, the first and second nucleic acid molecules are equimolar, or alternatively, one nucleic acid molecule is in excess compared to the other. Within other embodiments, the first and second nucleic acid molecules are of the same size or different sizes; the first nucleic molecule is a probe or a primer nucleic acid molecule and the second nucleic acid molecule is a target nucleic acid molecule or template for an extension product. If the first nucleic acid molecule is chimeric, then within one embodiment the probe does not contain a scissile linkage.

The concentration of RNase H and/or polyamine in the hybridization reaction mixture is dependent upon the quantity and type of nucleic acid molecules present in the sample. For example, within one embodiment preferred conditions for increasing the hybridization rates of two nucleic acid molecules using T thermophilus RNase H is a final concentration range between 5 x 10^"3 μM to 4.4 μM; for E. coli RNase H a final concentration range from 5 x 10^"2 μM to 50 μM; for spermine a final concentration range from 0.025 to 8.0 mM and for spermidine a final concentration range of 5 mM to 10 mM.

Within other embodiments, if RNase H is utilized for increasing the hybridization rates of the nucleic acid molecules wherein one of the nucleic acid molecules contains RNA as a scissile linkage, then a chelator is added to the mixture to prevent the cleavage of the RNA. Two representative chelators in this regard are ethylenediamine tetraacetic acid (EDTA) or ethylenebis(oxyethylenitrilo)-tetraacetic acid (EGTA). The more preferred chelator is EDTA in the range of 0.01 to 10 mM.

Stringency of the hybridization reaction of nucleic acid molecules may be controlled by use of salt, pH, temperature, solvent system and additives such as formamide. It is also expected that one skilled in the art will optimize the particular hybridization reaction as necessary. Standard hybridization mixtures typically contain monovalent salts (Na⁺ or K⁺) in excess of 100 mM. However, if such salt concentrations are utilized, they will decrease the acceleration rate that is observed with RNase H and polyamines. Thus, the preferred salt for use in the reaction mixture is Na⁺ and the concentrations of sodium phosphate buffer (PB) are as follows: for T. thermophilus RNase H, 2 to 100 mM PB; for E. coli RNase H, 2 to 20 mM PB; and for spermine, 0 mM to 100 mM PB (note that this range may be extended if higher concentrations of spermine are utilized).

The pH of the reaction mixture can also influence the hybridization rates in the presence of RNase H or polyamines and can range from pH 5 to 8. The preferred pH is from 5.5 to 7.5 and more preferred pH range is 6 to 7. A preferred buffer is N- tris[Hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES) or tris[hydroxy- methyljamino-methane (Tris). The more preferred buffer is 20 mM TES.

Reaction temperature can also influence the hybridization rates in the presence of polyamines or RNase H. Generally, it is preferred that thermostable RNase H be used in reactions occurring at high temperature. At low temperature non- thermostable RNase H can be used. The polyamine can be used at high and low temperatures. The hybridization reaction temperature can vary between 20°C and 70°C. The preferred temperature range is 15°C to 30°C below the melting temperature (T,_) of the duplex (temperature for half-strand separation). Detergents can be included in the reaction buffer and a preferred detergent is Triton X-100^® or Triton X-102^®. A more preferred detergent is Triton X- 100^®.

The acceleration of hybridization of nucleic acid molecules by this invention can be carried out in solution or immobilized formats. If the hybridization reaction is carried out with free nucleic acid molecules in a solution then the assay is termed in solution. If one of the nucleic acid molecules is bound to an insoluble matrix then the assay is termed immobilized. There are numerous ways to carry out hybridization assays and no attempt will be made to describe these in detail.

Representative hybridization assays for use in solutions include: measuring reannealing kinetics for genome complexity analysis; measuring hybridization kinetics for DNA:DNA, DNA:RNA, DNA: chimera oligonucleotide; detection of nucleic acid target with specific probe(s); preparation of differentially expressed genes or differentially represented genes; use of SI nuclease, RNase protection analysis or primer extension for measurement of abundance of specific mRNA molecules or mapping of 5' ends of mRNA, and heteroduplex mapping of mutations. Representative immobilized hybridization assays include: Southern or northern blot analysis and plaque or bacterial library screening. Insoluble matrices for binding nucleic acid include membranes, beads, micro-wells, gels, blots and slides or other solid supports. Other nucleic acid reactions that can likewise be accomplished utilizing the aforementioned methods include in situ hybridization, reverse transcription, primer extension, strand displacement, strand invasion, dideoxy sequencing, triplex formation and duplex recognition by nucleic acid molecules, antibodies or other ligands; amplification reaction such as PCR (Erlich (ed.) PCR Technology, Stockton Press, 1989; U.S. Pat. Nos. 4,683,195 and 4,683,202), ligase chain reaction (U.S. Pat. No. EP- A-320 308, WO 90/01069), strand displacement amplification (Walker et al, Nucleic Acids Res. 20:1691-1696, 1992), branched-chain DNA signal amplification (Urdea, Clin. Chem. 39:125-126, 1993), Q-beta-replicase (Lizardi et al., Biotechnology 6:1197- 1202, 1988), transcription-based amplification systems such as Transcriptional Amplification System (TAS, Kwoh et al., Proc. Natl. Acad. Sci USA 56:1173-1177, 1989), Self Sustained Sequence Replication (3SR, Guatelli et al., Proc. Natl. Acad. Sci USA 57:1874-1978. 1990), Nucleic Acid Sequence Based Amplification (NASBA, U.S. Pat. No. 5,409,818), Transcription-Mediated Amplification (TMA, U.S. Pat. No. 5,399,491) and 5' nuclease based amplification assays (Lyamichev et al., Science 260:778-783). Within certain embodiments of the invention, the hybridization reaction is a method other than polymerase chain reaction or ligase chain reaction.

The compositions and methods provided herein can also be utilized in a wide variety of other/related methods (e.g., U.S. Patent Nos. 5,210,015; 5,487,972; 5,422,253; 5,691,142; 5,719,028; 5,130,238; 5,554,517; 5,589,332, 5,480,784; 5,215,899; 5,169,766; 5,194,370; 5,474,916; 5,698,400; 5,656,430; and PCT publication nos. WO 88/10215; WO 92/08800; WO 96/02668; WO 97/19193; WO 97/09444; WO 96/21144; WO 92/22671).

E. Detection

After hybridization, the presence or absence of the nucleic acid molecule of interest in a sample can be detected with direct or indirect formats using various ligands, labels or tags that are well known in the art. Briefly, detection can be carried out with or without direct labeling of oligonucleotides and with or without a separation step for removing non-hybridized nucleic acid molecules.

Unlabeled nucleic acid molecules that are hybridized in solution can be detected by the physical changes that occur when single strand molecules form duplexes. The change in state can be detected by use of dsDNA intercalators (dyes) or antibodies. Examples of intercalators include ethidium bromide, YO-PRO-1 and SYBR Green I (cited in Ririe et al, Anal. Biochem. 245:154-160, 1997). Alternatively, hyperchromic and ultra violet spectrophotometric methods can be used. Another means is by detecting a change in the electrical conductivity when duplexes are formed.

Alternatively, one of the nucleic acid molecules can be labeled directly prior to hybridization, or indirectly with the use of a tag prior to hybridization and then attaching the label to the tag after hybridization. The labels can be, for example, radioisotopic, enzymatic, fluorescent, chemiluminescent, or bioluminescent. For use in solution or immobilized assays, the label can be attached directly or indirectly to one or both nucleic acid molecules. The various labels and formats are well known in the art and will not be discussed (see generally Keller and Manak, supra, Wetmur, supra).

Within yet other embodiments, nucleic acid molecules may be detected and solid support such as a bead (e.g., polymeric, metallic and/or magnetic), paper (e.g., nitrocellulose), or, a lateral flow device (e.g., strip, dipstick and the like see generally U.S. Patent Nos. 5,639,428; 5,635,362; 5,578,270; 5,547,861; 5,514,785; 5,457,027 5,399,500; 5,369,036; 5,260,025; 5,208,143; 5,204,061; 5,188,937; 5,166,054 5,139,934; 5,135,847; 5,093,231; 5,073,340; 4,962,024; 4,920,046; 4,904,583 4,874,710; 4,865,997; 4,861,728; 4,855,240; 4,703,017; and 4,847,194). Utilizing the method provided herein, hybridization rates can be increased or accelerated for both short and long nucleic acid molecules. The rates have also been increased in the presence of heterologous DNA. Further, the invention works under conditions of low salt in the hybridization reaction mixtures. An advantage of accelerating rate reactions in gene detection assays will be lower background, and thus, greater signal to noise ratio. Usually, in order for the hybridization reaction to be carried out in a short time, the hybridization reaction rates are probe driven. The higher the probe concentration, the faster the hybridization. But, when using probe labeled with a detectable moiety, the higher the probe concentration, the higher the background. Therefore, by reducing the concentration of the labeled probes and using RNase H and/or polyamines in the hybridization mixture to accelerate the rate, the reaction can be completed in a very short time with a concomitant corresponding reduction in background. Additionally the invention will allow for rapid hybridization under conditions of high stringency (low salts).

The following examples are offered by way of illustration, and not by way of limitation. EXAMPLES

EXAMPLE 1 CONSTRUCTION OF NUCLEIC ACID PROBES

Nucleic acid molecules can be synthesized utilizing standard chemistries on automated, solid-phase synthesizers such as PerSeptive Biosystems Expedite DNA synthesizer (Boston, MA), PE Applied Biosystems, Inc.'s Model 391 DNA Synthesizer (PCR-MATE EP) or PE Applied Biosystems, Inc.'s Model 394 DNA/RNA Synthesizer (Foster City, CA). Preferably, PerSeptive Biosystems Expedite DNA synthesizer is used and the manufacturer's modified protocol for making oligonucleotides is carried out.

Reagents for synthesis of oligonucleotides are commercially available from a variety of sources including synthesizer manufacturers such as PerSeptive Biosystems, PE Applied Biosystems Inc., Glen Research (Sterling, VA) and Biogenex. For DNA and RNA synthesis, the preferred fluorescein amidite, phosphoramidites of deoxy-and ribo-nucleosides, 2'-O-methyl and reagents, such as activator, Cap A, Cap B₄ oxidizer, and trityl deblocking reagent are available from PerSeptive Biosystems. Biotin-TEG-phosphoroamidite and Biotin-TEG-CPG are available from Glen Research. Ammonium hydroxide (28%) used for the deprotection of oligonucleotides is purchased from Aldrich. 1 M Tetrabutylammonium fluoride (TBAF) used for removing the 2'-O- tert-butyldimethylsilyl group is purchased from Aldrich and used after drying over molecular sieves for 24 hours. All buffers are prepared from autoclaved water and filtered through 0.2 μm filter. The following procedure is used for preparing biotinylated and/or fluoresceinated oligonucleotides. Biotin-TEG-CPG (1 μmol) is packed into a synthesis column. Nucleoside phosphoramidites are then linked to make the defined nucleic acid sequence using PerSeptive Biosystem's modified protocol for making oligonucleotides. Fluorescein-amidite is dissolved in acetonitrile to a final concentration of 0.1 M. The fluorescein amidite is loaded on the synthesizer and added to the 5'- end of the oligonucleotide. Alternatively, phosphoramidite containing thio-linker is added at the 5'- terminal of the chimeric probe using the modified protocol. After the deprotection step described below, the probe is purified by reverse phase HPLC using Millipore's R- 2 resin which retains the trityl containing oligonucleotide. In order to generate free reactive thio-group, the HPLC purified probe is treated with silver nitrate for 90 minutes at room temperature followed by neutralization of silver nitrate with dithiotheritol (DTT). The fluorescein-maleimide is then added to the free thio-group of the probe and then purified either by HPLC or by electrophoresis as described below.

After the synthesis of the oligonucleotide sequence, the resin bound oligonucleotide is treated initially with 25% ethanol-ammonium hydroxide (4 ml) at room temperature for 1 hour and subsequently at 55°C for 16 hours in a closed tube. The tube is cooled, supernant removed and concentrated to dryness in order to remove ammonia. The residue is dissolved in 1 ml of water and filtered through a 0.2 μm filter. The OD₂₆₀ is determined and an aliquot of approximately 2 OD₂₆₀ units is injected into the R-2 column of Biocad's HPLC to obtain a base line on the chromatogram for the tert-butyldimethylsilyl groups of the chimeric probe.

The remaining probe solution is lyophilized by centrifugal vacuum evaporator (Labconco) in a 1.5 ml microcentrifuge tube. The resulting oligonucleotide residue is deprotected with 1.0 M TBAF for 24 hours. To determine the extent of desilylation which has taken place, an aliquot of the TBAF reaction mixture is injected into the HPLC (R-2 column) using a linear gradient of 0 to 60% acetonitrile in 50 mM triethylammonium acetate (TEAA), pH 6.5. If only a partial desilylation has occurred, the TBAF reaction mixture is allowed to proceed for an additional 12 to 16 hours for complete removal of the protecting groups. The TBAF reaction mixture is quenched with 100 mM NaOAc, pH 5.5 and evaporated to dryness. The crude oligonucleotide product is desalted on a P-6 column (2 cm x 10 cm, Bio-Rad), the fractions are concentrated to approximately 1 ml and the concentration measured at OD₂₆₀.

The crude oligonucleotide is purified by polyacrylamide gel electrophoresis (PAGE) using 20% polyacrylamide-7 M urea. The running gel buffer is 1 x TBE (Tris-Borate- ethylenediamine tetraacetic acid (EDTA), pH 8.3 ) and the electrophoresis is carried out at 50 mA current for 3.5 to 4 hours. The oligonucleotide band is visualized with UV light, excised, placed in a 15 ml plastic conical tube and extracted by crushing and soaking the gel in 5 ml of 50 mM NaOAc (pH 5.5) for approximately 12 hours. The tubes are then centrifuged at 3000 RPM and the supernatant carefully removed with a Pasteur pipette. The gel is rinsed with 2 ml of the extraction buffer to remove any residual product. The combined extract is concentrated to a volume of approximately 1 ml and desalted on a P-6 column. The fractions containing the probe are pooled and concentrated to a final volume of approximately 2 ml. The analytical purity of oligonucleotides is checked by labeling the 5'- end of oligonucleotide with [γ^Pj-ATP and T4-polynucleotide kinase and then running the labeled oligonucleotide on PAGE. OD₂₆₀ is measured using Hewlett Packard's 845X UV spectrophotometer. The oligonucleotide solution is filtered through a 0.2 μm filter and stored at -20°C.

Utilizing the above procedure, the following oligomers below are synthesized. In these sequences, upper case letters have been utilized to denote deoxyribonucleotides, lower case letters to denote ribonucleotides, and underlined letters to denote 2'-O-methyl linkages.

mecA945-29D (SEQ ID NO: 1)

5'-AAT AGA GAA AAA GAA AAA AGA TGG CAA AG-3'

mecA945T (SEQ ID NO:2)

5'-CTT TGC CAT CTT TTT TCT TTT TCT CTA TT-3'

mecA932-59R (SEQ ID NO:3)

5'-cgc aca uac auu aau aga gaa aaa gaa aaa aga ugg caa aga uau uca acu aac uau ug-3'

ccmecA915-89 (SEQ ID NO:4)

5'-GAA CTT TAG CAT CAA TAG TTA GTT GAA TAT CTT TGC CAT CTT TTT

TCTTTT TCT CTATTAATG TAT GTGCGATTGTAT TGC TAT TAT CG-3'

mecA945-29[P] (SEQ ID NO:5)

5'-AAT AGA GAA AAA Gaa aaA AGA TGG CAA AG-3'

mecA945-29A₁₈P (SEQ ID NO:6)

5'-AAT AGA GAA AAA Gaa aaA AGA TGG CAA AGA₁₈-3'

mecA945-29(2' OMe) (SEQ ID NO:7)

5'-AAT AGA GAA AAA Gaa aaA AGA TGG CAA AG-3'

mecA945-29R (SEQ ID NO:8) 5'- aau aga gaa aaa gaa aaa aga ugg caa ag-3' mecA834-25 (SEQ ID NO:9)

5'-TGG TAA AAA GGG ACT CGA AAA ACT T-3'

mecAL1039-22 (SEQ ID NO: 10) 5'-GGT GGA TAG CAG TAC CTG AGC C-3'

mecA869-29 (SEQ ID NO: 11)

5'-AGC TCC AAC ATG AAG ATG GCT ATC GTG TC-3'

mecAL1042-30 (SEQ ID NO:12)

5'-ACC TGT TTG AGG GTG GAT AGC AGT ACC TGA-3'

The oligonucleotides in the following examples are 5' labeled with radioactive [³²P]-ATP as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989, using T4 polynucleotide kinase (RTG; Pharmacia Biotech Inc., Piscataway, N . (Pharmacia)). The labeled probe is purified from non-incorporated [³²P]-ATP by G50 NICK column (Pharmacia) chromatography.

EXAMPLE 2

PREPARATION OF THERMOSTABLE RNASE H

Cloning of a thermostable RNase H and its expression can be accomplished following procedures described in in WO 95/05480 and Bekkaoui et al., BioTechniques 20:240-248, 1996. Briefly, the T. thermophilus RNase H gene (Kanaya & Itaya, J. Biol. Chem. 267:10184-10192, 1992) is cloned by PCR into vector pT7-7 (pIDB9) and is subcloned into the vector pETl la (Novagen) resulting in the plasmid pIDB33. Plasmid pIDB33 is subsequently transformed into the bacterial strain BL21(DE3) (Novagen, Madison, WI). BL21(DE3) cells containing pIDB33 are grown at 37°C in LB medium (Sambrook et al, 1990) containing 0.1 mg/ml ampicillin. When the culture is at an OD₆₀₀ of 0.6 - 0.8, IPTG is added to a final concentration of 0.5 mM and the cells are cultured for four more hours. RNase H is expressed in the inclusion bodies with the pIDB33 construct.

Cells are harvested by centrifugation at 3000x g for 15 minutes at 4°C. Cell pellets are resuspended at 1 g fresh weight in 5 ml of TE buffer (10 mM Tris, pH 7.4, 1 mM ethylenediamine tetraacetic acid (EDTA) buffer). The cells are lysed on dry ice/ethanol bath using a sonicator (Branson, model 450) and centrifuged at 15,000x g for 30 minutes at 4°C. The pellet is resuspended in 7 M urea in TE buffer, pH 8.0 and incubated with stirring for 2 hours at 4°C. The resuspended cells are sonicated for 2 minutes on ice, followed by centrifugation at 12,000x g for 10 minutes and the supernatant is collected and dialyzed overnight against 1 1 of urea sodium acetate buffer (8 M urea, 20 mM sodium acetate, pH 5.5) with two changes. After a centrifugation for 20 minutes at 31,000x g, the clear protein supernatant solution (150 ml) is collected and mixed with approximately 25 ml of pre-swollen phosphocellulose (equilibrated 2 x in column buffer, PI 1, Whatman International Ltd., Kent, UK) for 3 hours. The resulting slurry is washed twice with the urea sodium acetate buffer and poured into a column. The column is connected to an FPLC system (Pharmacia) and step washed twice with 140 mM and 210 mM NaCl in the urea sodium acetate buffer. The protein is then eluted using a 0.21 to 0.7 M NaCl linear gradient in the urea sodium acetate buffer. At the end of the salt gradient, the column is maintained at 0.7 M NaCl until all the protein is eluted. Fractions are analyzed by SDS-PAGE and those containing RNase H are pooled and desalted using a Sephadex G-25 column with buffer containing 150 mM NaCl in 20 mM sodium acetate, pH 5.5. The eluted protein fractions are pooled, concentrated with a Centriprep 10 filter (Amicon, Beverly, MA), and stored at -20°C in glycerol storage buffer (40% glycerol, 150 mM NaCl and 20 mM sodium acetate, pH 5.5).

EXAMPLE 3 PURIFICATION OF GENOMIC STAPHYLOCOCCUS A UREUS DNA

The following example describes the procedure for purification of genomic DNA from methicillin resistant S. aureus isolates (MRSA, isolates ATCC 33592, American Type Culture Collection, Rockville, MD) and methicillin sensitive S. aureus (MSSA, ATCC 11632, American Type Culture Collection) (see generally Marmur, e.λ. Enzymol. 100:126-138, 1989).

Briefly, a pre-culture of S. aureus cells is grown in 40 ml of trypticase soy broth (TSB) for 6 to 8 hours at 37°C. The pre-culture is then added to 1 liter of TSB and grown overnight at 37°C with shaking. The cells are pelleted and washed once with 140 mM NaCl, 20 mM EDTA and 50 mM Tris, pH 8.0 (TSE) buffer at 6800x g (Sorvall) for 5 minutes at 5°C to 10°C. Lysis of cells is carried out by addition of 0.014 mg/ml lysostaphin (Sigma Chemical Company, St. Louis, MO) and 0.007 mg/ml lysozyme (Sigma) and the mixture incubated at 37°C for 1 hr with shaking. Sodium dodecyl sulfate (SDS, 20%, electrophoretic grade) is added to a final concentration of 0.09%, and the suspension is mixed and incubated in a water bath at 50°C to 60°C for 10 minutes and held at room temperature for 1 hour. The lysate is then cooled to room temperature, followed by addition of 24 ml of 5 M NaClO₄ and 40 ml of 25:24:1 of phenol:chloroform:isoamyl alcohol (v/v, PCIAA), and shaken for 2 hours at room temperature. The emulsion is aliquoted into sterile 30 ml glass tubes (Corex) and phase separation is carried out by centrifugation at 5000 rpm for 5 minutes in a table top centrifuge (Eppendorf). The upper phase, containing the nucleic acid, is collected and precipitated by layering with 2 volumes of 95% ethanol. This is followed by spooling of the crude genomic DNA with a sterile glass rod, and resuspension in 40 ml of sterile 15 mM NaCl, 1.5 mM trisodium citrate (O.lx SSC) buffer. Remaining RNA is degraded by addition of RNase A solution (2 mg/ml, Pharmacia), to a final concentration of 50 μg/ml and RNase Tl (2500 units/ml, Gibco BRL Life Technologies, Gaithersburg, MD) to a final concentration of 15 units/ml, to the crude DNA solution and incubating for 3 hours at 37°C.

For removal of any remaining proteins, 2 ml of SDS (20%) and 2 ml of Proteinase K (5 mg/ml, Gibco BRL) are added and the solution is incubated at 50°C for 5 minutes followed by a 30 minutes incubation at room temperature. The above PCIAA treatment is repeated with a 20 minutes mixing, followed by centrifugation, precipitation of the aqueous layer with ethanol, and spooling of DNA as described above, with the final resuspension in 10 ml of O.lx SSC. The solution can be left overnight at this stage or processing continued by addition of 1 ml of lOx SSC to bring final concentration to lx SSC, and 10 ml of chloroform-isoamyl alcohol (24:1, v/v, CIAA) with shaking for 15 minutes.

The solution is then aliquoted into glass tubes and centrifuged at 5000 rpm for 5 minutes for phase separation. The lower organic phase is removed and the aqueous phase with the interface is re-extracted as described above with CIAA until there is minimal protein at the interface. This is followed by removal of the aqueous layer, precipitation with ethanol, and DNA spooling as described previously. The DNA is resuspended in 5 ml of 0.01 x SSC and can be stored overnight. The DNA is then dialyzed with one buffer change against 0.01 x SSC at 4°C over a period of 4 hours, repeated once overnight, and then repeated once again for a further 4 hours. The amount of purified genomic DNA can be determined by UV spectrophotometry and then sonicated (Branson, model 250/450) for 10 minutes to reduce the size of DNA to less than or equal to 1000 base pairs (bp).

EXAMPLE 4 BINDING ASSAY FOR DETERMINING HYBRIDIZATION RATES OF NUCLEIC ACIDS

This example describes an assay for determining the hybridization rates of nucleic acids using hydroxylapatite (see generally Britten et al., Methods. Enzymol. 29:363-418, 1974 and US Patent No. 5,132,207). Briefly, oligonucleotides are first synthesized and then labeled with ³²P as described in Example 1. Thermostable RNase H is prepared as described in Example 2 and E. coli RNase H is prepared as described by Kanaya and Crouch, J. Biol. Chem. 258:1276- 1281, 1983. Spermine and spermidine are obtained from Sigma Chemical Company and hydroxylapatite (HAP, high resolution) is from Calbiochem (La Jolla, CA). Prior to the assay, the unlabeled excess oligonucleotide and labeled oligonucleotide are preincubated at 40°C. Unless specified otherwise, the final hybridization reaction mixture contains the test compound(s), unlabeled excess oligonucleotide, labeled oligonucleotide, 0.05% Triton X-100^®, 1.0 mM EDTA, specified salt as NaCl or sodium phosphate buffer (PB, equimolar monobasic and dibasic, pH 6.8), and 20 mM N-tris[Hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES, Sigma).

Oligonucleotides are mixed and timing started immediately upon addition of the accelerator being tested. An aliquot is removed from the reaction mixture at specified time intervals after the start of reaction. The sample is diluted 10 fold in HAP buffer (0.01% SDS and 100 mM PB) and passed over a column of pre- equilibrated HAP at 40°C. Non-hybridized single stranded oligonucleotides are removed from the column by passing PB buffer over the column. The oligonucleotides bound to HAP are recovered by dissolving the HAP in 6 N HC1. The flow through fraction from the column and the bound fraction (back peak) from the dissolved HAP are assayed for ³²P-labeled activity using Cerenkov counting (Berger & Krug, BioTechniques, :38-46, 1985).

The zero time binding, or background binding is determined and subtracted from the back peak (Britten et al., supra; Wetmur, supra). This yields the percentage double strand formed in the hybridization reaction at the specified time. Hybridization reactions which are carried out with excess non-labeled oligonucleotide to labeled oligonucleotide follow pseudo-first order kinetics and thus can be analyzed as described by Wetmur, supra. Hybridization rates (k_a) and half time of the reaction (t_1/2) are obtained from the data of percentage double strand formation over time from the HAP binding assay.

EXAMPLE 5

ACCELERATION OF DNA:DNA, DNA:RNA AND DNA:CHIMERA

HYBRIDIZATION USING THERMOSTABLE RNASE H

The following example demonstrates the impressive acceleration of

DNA:DNA, RNA:DNA and DNA:Chimeric oligonucleotide hybridization in the presence of T. thermophilus RNase H.

Briefly, the hybridization rates of different types of oligonucleotides are analyzed in reaction mixtures containing: (i) ten times excess unlabeled oligonucleotide to labeled oligonucleotide in 20 mM PB (sodium phosphate); (ii) ten times excess unlabeled oligonucleotide to labeled oligonucleotide in 20 mM PB with T. thermophilus RNase H; and (iii) hundred times excess unlabeled oligonucleotide to labeled oligonucleotide in 100 mM PB, is determined.

For these experiments, the types of nucleic acid hybridization are specified in Column 1 of Table 1, the unlabeled and labeled single stranded oligonucleotides are specified in Columns 2 and 3. The experiments for measuring the hybridization rates using HAP binding assay were carried out essentially as described in Example 4 with the following exceptions:

(1) The final concentrations in the hybridization reaction mixture testing the effect of RNase H using ten times excess unlabeled oligonucleotide and low salt, were as follows: 1.0 x 10^"n M unlabeled oligonucleotide, 1.0 x 10^"12 M labeled oligonucleotide, 0.5 μM RNase H, 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM sodium phosphate and 20 mM TES. All listed combinations of nucleic acid hybridization (Table 1, Columns 2 & 3) were tested. Each reaction mixture was sampled by HAP for percentage double strand binding at the following time intervals: 15 seconds, 30 seconds, 45 seconds, 1 minute and 2 minutes.

(2) The final concentrations in the hybridization reaction mixtures testing the effect of 10 times excess unlabeled oligonucleotide and low salt were as follows: 1.0 x 10 " M unlabeled oligonucleotide, 1.0 x 10^"12 M labeled oligonucleotide, 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM sodium phosphate and 20 mM TES. Only the specified DNA:DNA and chimera:DNA hybridizations were carried out and each reaction mixture were sampled for HAP analyses as follows: 0, 30 minutes, 60 minutes, 90 minutes and 120 minutes.

(3) The final concentrations in the hybridization reaction mixtures testing the effect of 100 times unlabeled oligonucleotide concentration and high salt in the hybridization reaction mixtures, were as follows: 5.0 x 10 ¹⁰ M unlabeled oligonucleotide, 5.0 x 10 ¹² M labeled oligonucleotide, 0.05% Triton X-100^®, 100 mM sodium phosphate, 1.0 mM EDTA and 20 mM TES. All listed combinations of nucleic acid hybridization (Table 1, Columns 2 & 3) were tested and sampling times for HAP analyses were as follows: 30 seconds, 8 minutes, 16 minutes, 30 minutes and 60 minutes.

TABLE 1. Type of nucleic acid hybridization and the labeled and unlabeled oligonucleotide sequence names and SEQ ID NOS.

Results

From the above experimental results the rate measurements (k_a), t_1/2 and rate acceleration (R) are calculated and presented in Table 2. The hybridization rates in column 4 have been normalized to salt concentrations of 20 mM PB in order to compare the results of rates using different salt conditions (Britten et al., supra). Briefly at 20 mM PB and 10 times excess unlabeled oligonucleotides, there was no detectable hybridization between the DNA:DNA and the chimera:DNA oligonucleotides over the time period of the experiment (2 hours; Table 2, Column 2). As expected, the hybridization rates increased with the use of 100 times excess unlabeled oligonucleotide and 100 mM PB in the reaction mixture. The rates of the different types of oligonucleotides ranged from 4.6 x 10² M^"1 s^"1 to 1.9 x 10³ M^"1 s^"1 (Table 2, Column 4). However, with the use of thermostable RNase H there was an unexpected and surprising increase in the hybridization rates of all the tested oligonucleotides ranging from 2.9 x 10⁸ M^"1 s^"1 to 1.4 x 10⁹ M^"1 s^"1. (Table 2, Column 3). To place this in perspective, the t,_/2 for the reaction mediated by RNase H , ranged from 0.6 minutes to 4 minutes. This was in contrast to time period of greater than 400 days under conditions of 100 times excess oligonucleotide for driving the reaction (Table 2, Columns 3 and 4). The relative RNase H mediated accelerations were estimated to be lO³ to 10⁶ -fold faster compared to the use of 100 times excess unlabeled oligonucleotides (Table 2, Column 5, rates adjusted to 20 mM PB for comparison). The above example demonstrates that thermostable RNase H can rapidly accelerate hybridization rates of chimeric, non-chimeric and different size of nucleic acid molecules and the reactions can be carried out with half time of less than 4 minutes.

TABLE 2. Comparison of nucleic acid hybridization rates under different conditions

t

m r co

Please refer to Table 1 for the specific sequence names and SEQ ID NOS

U-ODN L-ODN refers to the ratio of unlabeled oligonucleotide labeled oligonucleotide used in the experiment ' R is the k_a of RNase H mediated (Column 3)/ k_a of 100 times excess oligonucleotide mediated (Column 4) reactions ^*" hybridization rates (kj are normalized to 20 mM sodium phosphate (equivalent to 30 mM NaCl) ^*** not detected over the experimental period (2 hours) n/d - not done

EXAMPLE 6 ACCELERATION OF CHIMERIC ANALOG OLIGONUCLEOTIDE:DNA HYBRIDIZATION USING

THERMOSTABLE RNASE H

The following example demonstrates T. thermophilus RNase H mediated acceleration of a 2'-O-Methyl RNA chimeric oligonucleotide:DNA hybridization.

The effect of T. thermophilus RNase H on the hybridization rate of oligonucleotides mecA945-29(2' OMe) (SEQ ID NO:7) and mecA945T (SEQ ID NO:2) was examined. The oligonucleotides were synthesized and labeled as described in Example 1 and T. thermophilus RNase H was prepared as described in Example 2. The reaction conditions were identical to those described in Example 4 except that the final concentrations in the hybridization reaction mixture were as follows: 1.0 x 10^"n M mecA945-29(2' OMe) unlabeled oligonucleotide, 1.0 x 10 ¹² M labeled oligonucleotide mecA945T, 0.5 μM RNase H, 0.05% Triton X-100^®, 1.0 mM EDTA, 1.65 mM Na⁺ (equivalent to 1.1 mM PB) and 20 mM TES. The reaction mixture was sampled at the following intervals: 15 seconds, 30 seconds, 45 seconds, 60 seconds and 5 minutes.

Under the above experimental conditions, T. thermophilus RNase H mediated hybridization rate for the analog chimera:DNA was found to be 2.8 x 10⁹ M^"1 s^"1. This rate is comparable to the high rates observed with other nucleic acid molecules mediated by T. thermophilus RNase H in Example 5.

Therefore, the above experiment shows that thermostable RNase H can be used to accelerates hybridization rates of nucleic acid molecules with modified and non-modified linkages.

EXAMPLE 7 ACCELERATION OF DNA:DNA HYBRIDIZATION USING NON-THERMOSTABLE RNASE H

The following example demonstrates the acceleration of DNA:DNA oligonucleotide hybridization mediated by E. coli RNase H.

The effect of E. coli RNase H on the hybridization rate of oligonucleotides mecA945-29D (SEQ ID NO:l ) and its complementary target mecA945T (SEQ ID NO:2) was examined. The oligonucleotides were synthesized and labeled as described in Example 1 and E. coli RNase was prepared by the method of Kanaya and Crouch, J. Biol. Chem. 255.T276-1281, 1983. The reaction conditions were identical to those described in Example 4 except that the final concentrations in the hybridization reaction were as follows: 1.0 x 10^'9 M unlabeled mecA945-29D oligonucleotide, 2.0 x 10^"n M labeled oligonucleotide mecA945T, 0.5 μM RNase H, 0.05% Triton X-100^®, 1.0 mM EDTA, 3 mM Na⁺ (equivalent to 2 mM PB) and 20 mM TES. The reaction mixture was sampled at the following intervals: 30 seconds, 10 minutes, 20 minutes, 30 minutes and 60 minutes.

Under the above experimental conditions, E. coli RNase H mediated hybridization rate was calculated to be 1.6 x 10⁶ M^"1 s^"1.

The above experiment shows that non-thermostable RNase H can be used to increase hybridization rates of complementary nucleic acid molecules.

EXAMPLE 8 ACCELERATION OF DNA:DNA OLIGONUCLEOTIDE HYBRIDIZATION WITH SPERMINE OR T.

THERMOPHILUS KNASE H, IN THE PRESENCE OF HETEROLOGOUS DNA

The following example demonstrates the use of a polyamine and/or RNase H for rapid acceleration of DNA:DNA hybridization even in the presence of heterologous DNA.

The effect of spermine and/or T. thermophilus RNase H on the hybridization rates of complementary synthetic sequences from methicillin resistant S. aureus, unlabelled mecA945- 29-D (SEQ ID NO:l) to labeled mecA945-29T (SEQ ID NO:2), were examined in the presence or absence of genomic methicillin sensitive S. aureus (MSSA) DNA. The oligonucleotides were synthesized and labeled as described in Example 1, T. thermophilus RNase H was prepared as described in Example 2 and genomic MSSA DNA was prepared as described in Example 3. The experiment was essentially carried out and analyzed as described in Example 4 with the following exceptions:

(1) For the experiment testing the effect of RNase H on hybridization rate in the presence of MSSA DNA, the final concentrations in a hybridization reaction volume of 100 μl were as follows: 3.6 x 10^_u M of unlabeled mecA945-29D, 1 x 10^"12 M of labeled mecA945T in 0.05% Triton X-100^® , 1.0 mM EDTA, 20 mM TES, 105 ng/100 μl of MSSA, 1.65 μM RNase H and 5.6 mM Na⁺ (equivalent to 3.7 mM PB). The sampling times for HAP binding analysis were: 15 seconds, 30 seconds, 1 minute[s], 2 minutes, 10 minutes.

(2) For the experiment testing the effect of spermine in the presence or absence of MSSA DNA, the final concentrations in a hybridization reaction volume of 100 μl were as follows: 3.6 x 10^"" M of unlabeled mecA945-29D, 1 x 10^"12 M of labeled mecA945T in 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM TES, 105 ng MSSA, 1.0 mM spermine and 5.6 mM Na⁺ (equivalent to 3.7 mM PB). The sampling times for the HAP analysis were the same as above (1).

Table 3 summarizes the results of the above experiment. Briefly, in the absence of spermine or RNase H there was no observable hybridization of DNA:DNA oligonucleotides in the presence or absence of heterologous MSSA DNA over the time course of the experiment. Inclusion of spermine in the reaction mixture caused a dramatic increase in the hybridization rates both in the absence or presence of MSSA DNA. T. thermophilus RNase H also increased the hybridization rates, as previously observed, but the rate was lower in the presence of MSSA. The combination of spermine and RNase H in the reaction mixture also led to increased hybridization rates in the presence or absence of MSSA. This translates to a decrease in the reaction time for the hybridization reaction. Therefore the t_1/2 decreased from being unmeasurable during the time course of the experiments in the absence of spermine and RNase H to 0.6 minutes when both spermine and RNase H are used in the presence of heterologous DNA.

The above example demonstrates that the polyamine spermine, RNase H or a combination of both, can be used to accelerate hybridization of complementary nucleic acid molecules even in the presence of heterologous nucleic acid molecules.

Table 3. Effect of spermine and T. thermophilus RNase H on the hybridization rates of mecA945- 29D:mecA945T oligonucleotides with background of heterologous methicillin sensitive S. aureus (MSSA) DNA

^* hybridization rates are at Na⁺ιon equivalent to 3.7 mM PB ^** - indicates absence of additive m the hybridization reaction mixture ^***+ indicates presence of additive in the hybridization reaction mixture ^**** nd indicates not detected over the time period of the experiment EXAMPLE 9

CONCENTRATION RANGE OF SPERMINE AND RNASE H FOR

DNA:DNA DUPLEX FORMATION

The following example shows the range of spermine, E. coli and T. thermophilus RNase H concentrations for maximum percentage double strand formation of DNA oligonucleotides. The experiments were carried out using HAP binding assay as described in Examples 4 with the following exceptions:

(1) Spermine concentration range tested was from 0.025 mM to 8.0 mM (final concentration) in the hybridization reaction mixture which contained the following: 1.0 x 10^"11 M unlabeled oligonucleotide mecA945-29D (SEQ ID NO:l), 2 x 10^"12 M labeled oligonucleotide mecA945T (SEQ ID NO:2), 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM TES buffer and 3 mM Na⁺ (equivalent to 2 mM PB). The sampling time for HAP back peak analysis was carried out at 8 minutes. The back peak, an indication of double strand formation, was used for all the comparisons.

(2) T. thermophilus RNase H concentration range tested was from 5 x 10^"3 to 4.4 μM with the same hybridization conditions as in (1) above with the exception that RNase H was substituted for spermine.

(3) E. coli RNase H concentration range tested was from 5 x 10^'13'- to 50 μM, using the same conditions as in (1) above with the exceptions that E. coli RNase H was substituted for spermine, the labeled oligonucleotide was mecA945-29, the unlabeled oligonucleotide was at 1 x 10^"8 M, and the sampling time was 10 minutes.

The results of the above experiments showed that all the spermine concentration tested mediated accelerated double strand formation. In particular the concentration from 0.1 mM to 8.0 mM resulted in 70.9% to 83.8% back peak. T thermophilus RNase H concentration range from 0.01 μg/100 μl to 8.8 μg/100 μl (5 x 10^"3 to 4.4 μM) resulted in 15.1% to 85% back peak. For E. coli RNase H concentration ranging from 0.1 μg/100 μl to 10 μg/100 μl (5 x 10^"2 to 50 μM), resulted in 7.8% to 25.4% back peak.

The above example shows that each of the accelerators tested had a different concentration range for double strand formation in a hybridization reaction under the above conditions. EXAMPLE 10 ACCELERATION OF DNA:CHIMERA HYBRIDIZATION WITH SPERMIDINE

The following example demonstrates that the polyamine spermidine accelerates hybridization rates of nucleic acid molecules.

The effect of spermidine on the hybridization rates of mecA945T (SEQ ID NO:2) and mecA945-29P (SEQ ID NO:5) oligonucleotides was tested. Initially a titration of spermidine was carried out to obtain the optimal concentration to use for the rate experiment. The oligonucleotides were synthesized and mecA945-29P was labeled as described in Example 1. The final spermidine concentration tested ranged from 0.01 mM to 100 mM. The reaction conditions were identical to those described in Example 4 except that the final concentrations in the hybridization reaction mixture were as follows: 1.0 x 10^"π M unlabeled mecA945T oligonucleotide, 1.0 x 10^"12 M labeled oligonucleotide mecA945-29P, 0.05% Triton X-100^®, 1.0 mM EDTA, 2 mM Na⁺ (equivalent to 3 mM PB) and 20 mM TES buffer. The sampling time for HAP back peak analysis was carried out at 8 minutes.

The maximum percentage back peak was observed with the concentration of spermidine from 5 mM to 10 mM.

Subsequently, 8 mM of spermidine was chosen to be used in the experiment for measuring hybridization rates. The experiment was carried out as described above except that the hybridization reaction mixture was sampled at the following intervals: 15 seconds, 6 minutes, 12 minutes, 18 minutes and 46 minutes. Using the above experimental conditions, spermidine mediated hybridization rate for the chimera:DNA oligonucleotide was calculated to be 4 x 10⁷ M^" ' s¹.

This example demonstrates that the polyamine spermidine can also be used to accelerate nucleic acid hybridization rates of nucleic acid molecules.

EXAMPLE 11 EFFECT OF SALT CONCENTRATION ON ACCELERATION OF HYBRIDIZATION WITH SPERMINE AND

RNASE H

The following example demonstrates that spermine, E. coli and T thermophilus RNase H mediated hybridization of nucleic acid molecules can take place in minimal salt conditions.

In particular, the oligonucleotides were synthesized and labeled as described in Example 1 , T thermophilus RNase H was prepared as described in Example 2 and E. coli RNase H was prepared by the method of Kanaya and Crouch, supra. Hybridization reactions and the HAP binding assays for examining the percentage double strand formation was essentially carried out as described in Examples 4, with the following exceptions:

(1) The final concentration in the hybridization reaction mixtures in the presence of E. coli or T. thermophilus RNase H, were as follows: 1.0 x 10^"8 M unlabeled mecA945-29D (SEQ ID NO:l), 2.0 x 10^"n M labeled mecA945T (SEQ ID NO:2), 0.5 μM RNase H, 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM TES buffer and salt concentration tested ranged from 2 mM to 100 mM sodium phosphate (final concentration). The sampling time for HAP back peak analysis was carried out at 30 seconds.

(2) The final concentration in the hybridization reaction mixtures in the presence of two concentrations of spermine were as follows: 1.0 x 10^"" M unlabeled mecA945T (SEQ ID NO:2), 2.0 x 10^"12 M labeled mecA945-29D (SEQ ID NO: l), 0.1 mM and 2 mM spermine, 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM TES buffer and salt concentration tested ranged from 0 mM to 100 mM sodium phosphate (final concentration). The sampling time for HAP back peak analysis was carried out at 30 seconds.

The results from this experiment showed surprising results in regards to the salt requirement in a hybridization reaction. The salt range for E. coli RNase H mediated hybridization ranged from 2 mM to 20 mM PB and the maximum percentage back peak occurred between 2 mM to 10 mM PB. In contrast to E. coli, T thermophilus RNase H salt range was observed to be broader and ranged from 2 mM to 100 mM with the maximum back peak between 2 mM to 20 mM PB. The salt range for 0.1 mM spermine was narrower than RNase H. The salt range was from 0 mM to 20 mM with the maximum back peak between 0 and 2 mM PB. Surprisingly, the salt range for spermine increased with the use of 2 mM spermine. The upper range for 2 mM spermine was greater than or equal to 100 mM PB.

The above example demonstrates that nucleic acid hybridization can take place under conditions that require no or minimal salt levels in the hybridization reaction mixture when polyamines or RNase H are present. It also shows that the salt range for spermine mediated acceleration reactions can be increased if the spermine concencentration is simultaneously increased.

EXAMPLE 12

ACCELERATION OF DNA:DNA HYBRIDIZATION USING THERMOSTABLE RNASE H IN THE

PRESENCE OF INCREASING CONCENTRATIONS OF HETEROLOGOUS DNA

The following example demonstrates that thermostable RNase H is capable of increasing hybridization rates of DNA:DNA complementary oligonucleotides in the presence of different concentrations of heterologous DNA.

The experiments described herein were carried out essentially as described in Example 4, with the following exceptions: 3.6 x 10^"u M unlabeled mecA945-29D (SEQ ID NO:l), 1 x 10^"12 M labeled mecA945T (SEQ ID NO:2), 1.65 μM RNase H, 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM TES buffer, 5.6 mM Na⁺ (equivalent to 3.7 mM PB). The MSSA concentration tested were as follows: 1.05 ng, 10.5 ng, 52.5 ng and 105 ng per 100 μl in a reaction volume of 1500 μl. Percentage double stranded nucleic acid was determined from the HAP binding assay by sampling at 30 seconds, 60 seconds, 120 seconds, 240 seconds, 480 seconds and the rate determined.

The following table (Table 4) summarizes the results of the above experiment. It shows that thermostable RNase H can increase hybridization rates in the presence of considerable amount of heterologous MSSA DNA. It was observed that for the samples containing 10.5 ng or less of MSSA DNA, the half-time for the reaction to be completed was similar to the control, i.e., with no added MSSA DNA, and as the concentration of MSSA DNA increased in the sample, the t_1/2 also increased.

From the above observations, it is hypothesized that RNase H in the reaction mixture may be binding to MSSA and therefore may not be available for accelerating the complementary hybridization as the concentration of MSSA increased in the sample.

Table 4. Effect of T thermophilus RNaseH on the hybridization rates of mecA945- 29D:mecA945-29T oligonucleotide in the presence of heterologous methicillin sensitive S. aureus (MSSA) DNA

k_a refers to the hybridization rates The above example demonstrates that thermostable RNase H can increase hybridization rates of complementary nucleic acid molecules in the presence of heterologous DNA and thereby decrease the time required for carrying out these reactions.

EXAMPLE 13 ACCELERATION OF EQUIMOLAR DNA:DNA HYBRIDIZATION USING THERMOSTABLE RNASE H OR

SPERMINE

The following example demonstrates that equimolar concentrations of DNA oligonucleotides in hybridization reactions can be rapidly accelerated by the use of T. thermophilus RNase H and spermine.

Briefly, synthetic oligonucleotides were synthesized as described in Example 1 and T thermophilus RNase H was prepared as described in Example 2. The experiment was carried out essentially as described in Example 4 and 5 with the following exceptions: 1 x 10^"10 M unlabeled mecA945T (SEQ ID NO:2), 1 x 10 ^!0 M labeled mecA945-29D (SEQ ID NO:l), 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM TES buffer, 3 mM Na⁺ (equivalent to 2 mM PB) and 0.5 μM RNase H or 2 mM spermine. The control, in the absence of RNase H or spermine, contained 500 times greater concentration of unlabeled and labeled oligonucleotides, i.e., 5 x 10^"8 M. Percentage double stranded nucleic acid was determined from the HAP binding assay and the hybridization rates were calculated using second order kinetics.

In the absence of spermine or RNase H, in the hybridization mixture, the rates of DNA:DNA hybridization was very slow, 8 x 10³ M^"'s^"'. In the presence of spermine or RNase H the rates increased to 2.7 x 10^s and 3.6 x 10⁸, respectively. This was approximately an acceleration of 10⁴ fold compared to the control.

This example demonstrates that rapid acceleration rates can be achieved using equimolar concentrations of nucleic acid molecules when mediated by spermine or RNase H.

EXAMPLE 14 ACCELERATION OF GENOMIC DNA:DNA REANNEALING USING THERMOSTABLE RNASE H OR

SPERMINE

The following example demonstrates that both T thermophilus RNase H and spermine accelerate the reannealing of denatured genomic DNA.

Briefly, T thermophilus RNase H was prepared as described in Example 2. MRSA was prepared as described in Example 3 and was random primer labeled (High Prime DNA Labeling Kit, Boehringer Mannheim). DNA was denatured at 95°C for 5 minutes, placed on ice and then incubated at 60°C prior to addition of RNase H or spermine.

The effect of 1 mM spermine, 1.65 μM RNase H or the combination of 1 mM spermine and 1.65 μM RNase H, on reannealing of 10 ng per 100 μl (1.5 x 10^"7 M bp) and 52 ng per 100 μl (8.0 x 10^"7 M bp) MRSA DNA were examined. The final hybridization reaction mixture contained the specified accelerator, specified concentration of MRSA DNA, 0.05% Triton X-100^®, 1.0 mM EDTA, 20 mM TES buffer, 5.6 mM Na⁺ (equivalent to 3.7 mM PB). Samples were taken at 0, 8 minutes, 16 minutes, 30 minutes and 60 minutes and the extent of double strand formation was determined using HAP analysis as described in Example 4. To facilitate rate measurements in the absence of spermine or RNase H, the MRSA DNA concentration was increased to 14,550 ng per 100 μl (3.7 x 10^"4 M bp) with the same conditions of salt as above or with an increase in salt concentration to 726 mM Na⁺ (equivalent to 484 mM PB). For 5.6 mM Na⁺ condition, samples were taken at the following intervals: 0 minutes, 33 minutes, 55 minutes, 150 minutes; and for the 726 mM Na⁺ condition, samples were taken at the following intervals: 0 minutes, 8min, 16 minutes, 30 minutes, 60 minutes. Reannealing rates were determined using second order kinetics.

Table 5 summarizes the results of the above experiment. Briefly, in the absence of RNase H or spermine the reannealing rates are extremely slow. Even when using high concentrations of DNA in the sample, i.e., 14,550 ng/lOOμl, the rate was so low that it was barely measurable. Increasing both the DNA concentration and the salt concentration, increased the rate to 2.0 M^"1 s^"1. However, the rates increased significantly with the addition of RNase H, spermine or a combination of RNase H and spermine, in the hybridization reaction mixture containing 10 and 52 ng/100 μl of MRSA DNA. The rate of increase was greater for spermine or spermine and RNase H combination, compared with RNase H by itself. When the concentration of MRSA was increased from 10 ng/100 μl to 52 ng/100 μl, the hybridization rate mediated by RNase H decreased, but this was not the case for spermine, under the above conditions. These results imply that the RNase H concentration may be limiting under condition of increased concentration of MRSA DNA and therefore, an increase in concentration of RNase H should theoretically increase the rates.

The above example demonstrates that spermine and thermostable RNase H or a combination of both can be utilized for increasing the reannealing rate of genomic DNA. Table 5. The effect of spermine, RNase H and combination of spermine and RNase H on the reannealing rates (k_a) of genomic methicillin resistant S. aureus DNA (MRSA)

Rates are not adjusted to same salt concentrations

EXAMPLE 15 SOUTHERN BLOT HYBRIDIZATION

The following example examines the utilization of spermine and low salt conditions for hybridization in Southern Blot analysis.

Briefly, probe mecA945-29D (SEQ ID NO: l) is synthesized and labeled as described in Example 1. MRSA and MSSA DNA is prepared as described in Example 3. Southern blot hybridizations are performed essentially as described by Sambrook et al., supra. Briefly, chromosomal DNA from MRSA and MSSA is electrophoresed on 1.5% agarose gel, and transferred to nitrocellulose membrane according to the standard procedure described generally in Maniatis et al. (supra). Hybridization with the probe is carried out using 0.1 mM to 2 mM spermine, and O.lx SSC or O.Olx SSC for times ranging from 0.5 hour to 3.5 hours. Membranes are washed twice with O.lx SSC (15 mM NaCl and 1.5 mM sodium citrate) and 0.1% SDS for 30 minutes and twice with O.lx SSC at room temperature for 30 minutes. The filter signal is analyzed on a Phosphorlmager™ using the IMAGEQUANT™ software (Molecular Dynamics, Sunnyvale, CA).

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMSWe claim:

1. A method of increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule, comprising: mixing a first nucleic acid molecule, a second nucleic acid molecule and a polyamine under conditions and for a time sufficient to permit hybridization of said first nucleic acid molecule to said second nucleic acid molecule.

2. The method according to claim 1 wherein said polyamine is spermine.

3. The method according to claim 1 wherein said polyamine is spermidine.

4. The method according to claim 1 wherein said first or second nucleic acid molecule is DNA .

5. The method according to claim 1 wherein said first or second nucleic acid molecule is RNA.

6. The method according to claim 1 wherein said first nucleic acid molecule or second nucleic molecule contains at least one phosphonate, diphosphonate, phosphorotriester, phosphoramidite, 2'-O alkyl or aryl ribonucleotide.

7. The method according to claim 1 wherein said first nucleic acid molecule is shorter than said second nucleic acid molecule.

8. The method according to claim 7, further comprising the step of extending said first nucleic acid molecule along said second nucleic acid molecule.

9. The method according to claim 1 wherein said first and second nucleic acid molecules are in solution.

10. The method according to claim 1 wherein said first nucleic acid molecule is bound to a solid support.

11. The method according to claim 10, further comprising the step of washing said solid support.

12. The method of claim 1, further comprising the step of passing said mixture through a polyacrylamide gel.

13. The method according to claim 1 wherein said first or second nucleic acid molecule is labeled.

14. The method according to claim 13, wherein said label is selected from the group consisting of a member of specific binding pair, chemiluminescence, fluorescent molecules, coenzymes, enzyme substrates and radioactive groups.

15. The method according to claim 1, wherein said mixture further comprises RNase H.

16. The method according to claim 15 wherein said RNase H is thermostable RNase H.

17. The method according to claim 15 wherein said RNase H is non- thermostable RNase H.

18. The method according to claim 2 wherein said mixture further comprises Na⁺ ion equivalent to 0 mM to 100 mM sodium phosphate.

19. The method according to claims 15 wherein said mixture further comprises Na⁺ ion equivalent to 2 mM to 100 mM sodium phosphate.

20. The method according to claim 1, wherein said mixture further comprises a chelator.

21. The method according to claim 20, wherein said chelator is EDTA or EGTA.

22. A method of increasing the hybridization rate of a first nucleic acid molecule to a second nucleic acid molecule, comprising: mixing a first nucleic acid molecule, a second nucleic acid molecule and RNase H under conditions and for a time sufficient to permit hybridization of said first nucleic acid molecule to said second nucleic acid molecule, with the proviso that said first and second nucleic acid molecule does not contain scissile linkage, and, with the further proviso that if said first nucleic acid is DNA, that the second nucleic acid molecule is not RNA, or if the first nucleic acid is RNA, the second nucleic acid molecule is not DNA.

23. The method according to claim 22 wherein said RNase H is thermostable RNase H.

24. The method according to claim 23 wherein said RNase H is non- thermostable RNase H.

25. The method according to claim 23 wherein said first or second nucleic acid molecule is DNA .

26. The method according to claim 23 wherein said first or second nucleic acid molecule is RNA.

27. The method according to claim 23 wherein said first nucleic acid molecule or second nucleic molecule contains at least one phosphonate, diphosphonate, phosphorotriester, phosphoramidite, 2'-O alkyl or aryl ribonucleotide.

28. The method according to claim 23 wherein said first nucleic acid molecule is shorter than said second nucleic acid molecule.

29. The method according to claim 28, further comprising the step of extending said first nucleic acid molecule along said second nucleic acid molecule.

30. The method according to claim 23 wherein, said first and second nucleic acid molecules are in solution.

31. The method according to claim 23 wherein said first nucleic acid molecule is bound to a solid support.

32. The method according to claim 31, further comprising the step of washing said solid support.

33. The method of claim 23, further comprising the step of passing said mixture through a polyacrylamide gel.

34. The method according to claim 23 wherein said first or second nucleic acid molecule is labeled.

35. The method according to claim 34 wherein said label is selected from the group consisting of a member of specific binding pair, chemiluminescence, fluorescent molecules, coenzymes, enzyme substrates and radioactive groups.

36. The method according to claim 23, wherein said mixture further comprises polyamine.

37. The method according to claim 36 wherein said polyamine is spermine.

38. The method according to claim 36 wherein said polyamine is spermidine.

39. The method according to claim 23, wherein said mixture further comprises a chelator.