WO2001023618A2

WO2001023618A2 - Compositions and methods for reducing oligonucleotide hybridization and priming specificity

Info

Publication number: WO2001023618A2
Application number: PCT/US2000/026775
Authority: WO
Inventors: Jeffrey Van Ness; Lori K. Garrison
Original assignee: Qiagen Genomics Inc
Current assignee: Qiagen Genomics Inc
Priority date: 1999-09-30
Filing date: 2000-09-28
Publication date: 2001-04-05
Anticipated expiration: 2002-03-30
Also published as: WO2001023618A3; AU7733200A

Abstract

Annealing promoting compounds (APCs) are used to reduce the hybridization specificity of oligonucleotide duplexes containing one or more nucleotide mismatches as compared to perfectly base-paired oligonucleotide duplexes. These APCs are used in methods for the detection of target oligonucleotides that vary in nucleic acid sequence by one or more nucleic acid as compared to a control sequence.

Description

COMPOSITIONS AND METHODS FOR REDUCING OLIGONUCLEOTIDE HYBRIDIZATION AND PRIMING SPECIFICITY

TECHNICAL FIELD This invention relates to the fields of molecular biology, nucleic acid chemistry and cytogenetics and, more specifically, to compositions and methods to decrease the specificity of oligonucleotide hybridization and priming.

BACKGROUND OF THE INVENTION

Conventional methods for forming oligonucleotide duplexes require a high degree of nucleic acid sequence complementarity between the oligonucleotide probe or primer and the corresponding target oligonucleotide. Absent a high proportion of complementary nucleotides, a given oligonucleotide primer or probe will not hybridize to a specific nucleic acid target. While this situation is typically desirable and is relied upon in many assay systems, there are instances, as disclosed more fully herein, where hybridization (i.e., duplex formation) is desired between nucleic acid molecules having low complementarity. Thus, there exists an unmet need for compositions that reduce hybridization and priming specificity between a primer or probe and the target sequence, thereby permitting hybridization to occur with imperfectly base-paired oligonucleotide duplexes. Quite surprisingly, the present invention fulfills this and other related needs by providing compositions and methods that, inter alia, reduce the difference in discrimination temperature between a perfectly base-paired oligonucleotide duplex and an oligonucleotide duplex having one or more nucleotide mismatches.

SUMMARY OF THE INVENTION In one embodiment, the present invention provides a composition comprising an oligonucleotide and an annealing promoting compound (APC). The composition may be used, for example, to reduce the specificity of a hybridization reaction involving the oligonucleotide. The oligonucleotide may optionally be immobilized on a solid surface, where suitable solid surfaces include a nylon tip, a nylon bead, and a nylon membrane.

In a preferred embodiment, the APC is an aminoalcohol, where the aminoalcohol comprises at least one amine group and at least one hydroxyl group. The composition may further comprise an acid and/or a buffer, so that the aminoalcohol may be present in the composition, either entirely or in part, as a salt of the aminoalcohol. The composition is preferably aqueous, and has a pH of between 4 and 10. Exemplary aminoalcohol APCs are 4-hydroxypiperidine, l-methyl-3- piperidinemethanol, 4,4'-trimethylenebis(l-piperidineethanol), 3-piperidinemethanol, l-ethyl-4-hydroxy-piperidine, 2-piperidineethanol, 3-hydroxy-l-methylpiperidine, 1- ethyl-3-hydoxy-piperidine, 4-hydroxy- 1 -methylpiperidine, 1 -methyl -2- piperidinemethanol, 2-piperidine-methanol, 2,2,6,6-tetramethyl-4-piperidinol, l,4-bis(2- hydroxyethyl)piperazine and 1 -(2-hydroxyethyl)piperazine. In another embodiment, the present invention provides a method of decreasing the specificity of a hybridization reaction between two oligonucleotides. The method comprises adding an annealing promoting compound (APC) to a hybridization reaction between two oligonucleotides.

In another embodiment, the present invention provides a method of decreasing the specificity of a hybridization reaction between two oligonucleotides. The method comprises mixing a first oligonucleotide, a second oligonucleotide, and an annealing promoting compound (APC) under conditions suitable for the formation of an oligonucleotide duplex.

In another embodiment, the present invention provides a method of identifying a target oligonucleotide. The method comprises:

(a) mixing a first oligonucleotide having a sequence complementary to the target oligonucleotide, a second oligonucleotide having a sequence complementary to the complement of the target oligonucleotide, an annealing promoting compound (APC), a polymerase, a buffer compatible with polymerase activity, and a target oligonucleotide; (b) heating the mixture of (a) to a temperature above the melting temperature of the first oligonucleotide and the second oligonucleotide and their respective complementary sequences;

(c) reducing the temperature of the mixture of (b) to below the melting temperature, to thereby allow hybridization between the first oligonucleotide, the second oligonucleotide, and the target oligonucleotide;

(d) raising the temperature of the mixture of (c) to a temperature compatible with polymerase activity; and

(e) detecting a product of polymerization. These and other embodiments of the present invention will be apparent upon reference to the following detailed description and attached drawings. To this end, various references are set forth herein which describe in more detail certain procedures, compounds and/or compositions, and are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph illustrating thermal melt profiles of duplexes comprising either perfectly base-paired oligonucleotides or oligonucleotides having one base-pair mismatch. Percentage single-strand DNA (y-axis, fraction single stranded = α) is plotted versus temperature (°C, x-axis). The Ta of the duplex is the temperature at which 50% ofthe strands are in single-strand form. The helical to coil transition (HCT) is the temperature difference between an α of 0.2 (or 20% single-stranded) and an of 0.8 (or 80% single-stranded for a particular duplex). The temperature difference between two Tas at α = 0.5 define the ΔTa for two different duplexes.

Figures 2A-G are graphs illustrating thermal melt profiles for perfectly base-paired oligonucleotide duplexes and oligonucleotide duplexes having one base- pair mismatch. Thermal melt profiles were obtained in the presence of either 2- piperidine ethanol (2-PE, Figure 2A); 1 -methyl-2-piperidine methanol (MPM, Figure 2B); 1-piperidine ethanol (1-PE, Figure 2C); l-amino-2,6-dimethyl piperidine (AdmP, Figure 2D); SSC (Figure 2E); guanidinium isothiocyanate (GuSCN, Figure 2F); or PCR buffer (Figure 2G). Temperature is recorded in °C in each graph.

Figures 3A-D are graphs illustrating thermal melt profiles for oligonucleotide duplexes containing a varying percentage of G+C content. Thermal melt profiles were obtained in the presence of either 2-piperidine ethanol (2-PE, Figure

3A); SSC (Figure 3B); guanidinium isothiocyanate (GuSCN, Figure 3C); or PCR buffer

(Figure 3D).

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to an understanding thereof to define certain terms used herein.

The phrase "annealing promoting compound" (APC) refers to a substance that, when added to an oligonucleotide hybridization reaction, increases the difference in the helical coil transition (HCT) temperatures, decreases the difference in the discrimination temperatures (Ta) between a perfectly base-paired oligonucleotide duplex and an oligonucleotide duplex that contains one or more base-pair mismatch, and/or reduces the T of an oligonucleotide duplex from the T_d of the same oligonucleotide duplex in SSC. An APC may also be defined as a chemical that can decrease the van't Hoff enthalpy of an oligonucleotide duplex, preferably by at least 20%, when referenced to a standard salt solution (i.e., 0.165 M NaCl) when the APC is present in the hybridization reaction within a molarity range of 1 mM to 10 M.

The term "oligonucleotide" (ODN) refers to any polymer having two or more nucleotides. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, or an analog of either. See, e.g., PCT International Publication No. WO 98/13527 for suitable analogs, including abasic residues and specificity spacers. Oligonucleotide is used synonymously with the terms polynucleotide or nucleic acid. When used as probes or primers in hybridization reactions, preferred oligonucleotides are less than 100 nucleotides in length. More preferred are oligonucleotides that are less than 31 nucleotides. Still more preferred are oligonucleotides of less than 25 nucleotides. Most preferred are oligonucleotides of between 6 and 18 nucleotides in length. The phrase "base-pair mismatch" refers to all single and multiple nucleotide substitutions that perturb the hydrogen bonding between conventional base- pairs, e.g., G:C, A:T, or A:U, by substitution of a nucleotide with a moiety that does not hybridize to a corresponding nucleotide on the opposite strand of the oligonucleotide duplex. Such base-pair mismatches include, e.g., G:G, G:T, G:A, G:U, C:C, C:A, C:T, C:U, T:T, T:U, U:U, and A:A. Also included within the definition of base-pair mismatches are single or multiple nucleotide deletions or insertions that perturb the normal hydrogen bonding of a perfectly base-paired duplex. In addition, base-pair mismatches arise when one or both of the nucleotides in a base pair has undergone a covalent modification (e.g., methylation of a base) that disrupts the normal hydrogen bonding between the bases. Base-pair mismatches also include non-covalent modifications such as, for example, those resulting from incorporation of intercalating agents such as ethidium bromide and the like that perturb hydrogen bonding by altering the helicity and/or base stacking of an oligonucleotide duplex. The phrase "helical to coil transition" (HCT) refers to the difference between the temperature at which 80% of an oligonucleotide duplex is single-stranded and the temperature at which 20% of an oligonucleotide duplex is single-stranded.

The term "stringency" refers to the percentage of mismatched base-pairs that are tolerated for hybridization under a given condition. The phrase "discrimination temperature" (Ta) refers to the temperature at which 50% of an oligonucleotide duplex becomes single-stranded. Within the context of a hybridization reaction, T_d is the temperature that allows maximal discrimination between a perfectly base-paired ODN duplex and a duplex having one or more base- pair mismatches. The term "discrimination" refers to the difference in Ta (ΔT_d) between a perfectly base-paired oligonucleotide duplex and a duplex containing one or more base- pair mismatch.

The present invention provides compositions comprising a class of molecules, referred to herein as annealing promoting compounds (APCs), that, when added to an oligonucleotide hybridization reaction, decrease the discrimination between perfectly base-paired oligonucleotide duplexes and oligonucleotide duplexes containing one or more base-pair mismatches. Further provided are methods employing one or more of the inventive compositions that permit reduced discrimination between oligonucleotide duplexes. As described herein, the inventive compositions will find uses in methods in which it is advantageous to reduce oligonucleotide duplex hybridization specificity.

Thermodynamic Parameters Defining Oligonucleotide Duplexes and the Effect of APCs on Those Parameters

The hybridization of two complementary oligonucleotides to form a duplex may be described by a variety of thermodynamic parameters. The characteristic parameters of a thermal melting profile, under standard buffer conditions, for a perfectly base-paired oligonucleotide duplex and an oligonucleotide duplex having a single base-pair mismatch may be represented as shown in Figure 1. The range of temperatures over which a duplex transitions from 20% single- stranded (α = 0.2) to 80% single-stranded (α = 0.8) is termed the "helical to coil transition" or HCT and is expressed in °C. The temperature at which 50% of an oligonucleotide duplex melts into single strands is conventionally referred to as the melting temperature (T_m). Where, however, one oligonucleotide is attached to a solid surface, this temperature is referred to herein as the discrimination temperature (Ta). The stringency factor is the value of the slope (partial derivative) ofthe HCT at the T_d.

The difference in T_d for a perfectly base-paired oligonucleotide duplex and for a duplex having one or more base-pair mismatches (i.e., ΔT_d) is a measure of the ability of the target oligonucleotide to "discriminate" between the perfectly base- paired and mismatched oligonucleotide. The ΔT_d between a perfectly base-paired duplex and a duplex containing a mismatch is a function of the stringency factor or HCT of a given hybridization solution or APC. Comparison of melting profiles, obtained under standard buffer conditions, of a perfectly base-paired oligonucleotide duplex to that for an oligonucleotide duplex having one or more base-pair mismatches reveals that the mismatch causes a reduction in the melting temperature with a corresponding increase in HCT. Thus, ΔT_d decreases as the stringency factor decreases and HCT increases. The APCs of the present invention increase the ΔHCT and/or decrease ΔT_d (preferably bringing ΔT_d to zero) in an oligonucleotide hybridization reaction, and preferably achieve both effects. Transition enthalpies between a perfectly base-paired oligonucleotide duplex to two un-paired single strands can be calculated. See Breslauer, K.J., Chapter 15, "Methods for Obtaining Thermodynamic Data on Oligonucleotide Transitions," in Thermodynamic Data for Biochemistry and Biotechnology (ed. H. Hinz, Academic Press, New York, N.Y., 1986). The difference between a non-cooperative and cooperative transition is expressed in terms of ΔH_vH (van't Hoff enthalpy). In a cooperative transition, the value of (dα/dT)Ta is high, and therefore, the ΔH_vH is also high. In a non-cooperative transition, the value of (dα/dT)Ta is low, and therefore, the ΔH_VH is also low. (The term (dα/dT)Ta is the derivative of the slope of the melting curve at the Ta, α is defined as the fraction of total oligonucleotides in single stranded form, as plotted on the ordinate axis.)

The van't Hoff enthalpy for an oligonucleotide duplex can be determined from the differentiated equilibrium melting curve by plotting dα versus temperature. See Marky, L.A. and Breslauer, K.J., Biopolymers, 26(9): 1601 -1620, 1987. Briefly, thermodynamic data provide a basis for predicting the stability (ΔG') and temperature- dependent melting behavior (i.e., the helical to coil transition (HCT), (ΔH⁰)) from the primary sequence of bases in the duplex. A thermally induced helical to coil transition (from double strand to single strand) may be used to obtain values for the ΔH_vH. Analysis of the shape of the helical to coil transition is used to calculate the van't Hoff transition enthalpy. As described by Marky and Breslauer, supra, α is equal to the fraction of single strands in the duplex state. If α is plotted versus temperature, the temperature at which α takes the value of 0.5 is defined as the T_d. The equilibrium constant K for any transition can be expressed in the form of α, the van't Hoff enthalpy can be expressed as:

Δ-H_VH = RT2[dlnK/dT] or Δ-H_vH= -R[dlnK/d(l/T)] To solve the general expression when takes the value of 0.5 in terms of α the foregoing equation is differentiated and solved for at the T_d:

Δ-HVH = (2 + 2n)RT2(δα/dT)τ-τa which can also be written:

Δ-H_VH = (2 + 2n)R(δα/δ(l/T)_τ-τd It is assumed that a bi-molecularity exists where n=2 for the preceding equations and therefore the corresponding coefficient is equal to 6. Another assumption employed is that there is no dependence of T on concentration since at every temperature increment the concentration of single strands is zero (all unhybridized material is washed away from a solid support prior to the melting process and that at each 5°C temperature increment, the solid support is placed in a fresh solution; see the description, infra, on methods of obtaining thermal denaturation parameters). For any process at equilibrium, ΔG = -RT(lnKeq) and ΔG = ΔH - TΔS, and thus -RT (In K) = ΔH - TΔS.

It has been shown for bimolecular transitions that the full width or half- width of a differentiated melt curve at the half-height is inversely proportional to the van't Hoff transition enthalpy. See Gralla, J. and Crothers, D.M., J. Mol. Biol. 73:497-

511, 1973. As suggested, for an equilibrium of the form nA x> A_n the general forms of the van't Hoff equation are:

Δ-H_VH = B/((l/T,)-(l/T₂) (for the full width at half-height) Δ-H_VH = B 7((1/T_max)-(1/T₂) (for the upper half-width at half-height) where T_max is the temperature at the maximum, and Ti and T₂ correspond to the upper and lower temperatures at which value the change in the plotted temperature is equal to one-half of [(3α/d(l/T)_max]. For a molecularity of 2, -B = 10.14 and -B^'= 4.38. The detailed derivations are given in Marky and Breslauer, supra. This approach of measuring the van't Hoff enthalpies is particularly amenable to melting duplexes off solid supports as all problems associated with baselines and background are completely eliminated.

The equilibrium constant K for a helical transition of a molecularity of 2 can be expressed as the extent of α (the fraction of single strand molecules in a duplex). The value of K is usually determined at the T_m ofthe helical to coil transition where α = 0.5. This value of the T_m is then extrapolated to some reference temperature (e.g., 298K) using the empirically determined T_m (or T_d) and the calculated van't Hoff enthalpy (assumed to be temperature independent) and the integrated form of the van't Hoff equation: ln[K(T_m)/K(T_ref)] = ΔH°/R(l/T-1/T_m)

From the empirically determined value of K(T_ref), it is possible to determine ΔG° for the helical to coil transition using the relation ΔG° = ΔH° - TΔS⁰. Since the melting curves described here are concentration independent, the ln(K- _m) = 0 since K = 1 at the T_m. Therefore the van't Hoff equation reduces to: -In K(T) = ΔH°/R(l/T-1/T_m), which upon multiplying both sides by RT, provides

-RT InK(T) = ΔH°(1-T/T_m) = ΔG°

This expression can be used to calculate the transition free energy ΔG° at any temperature of interest (T) from the experimentally measured values of T_m and ΔH_VH- The corresponding ΔS° can be calculated from relation ΔG° = ΔH° - TΔS⁰.

APCs

As a consequence of increasing the ΔHCT of an oligonucleotide duplex, an APC decreases the stringency factor of a hybridization solution or solvent, where the stringency factor is the value of the slope (partial derivative) of the helical to coil transition at the value of the Ta. As discussed above, the stringency factor can be used to identify an APC.

Several consequences flow from the addition of an APC to an oligonucleotide hybridization reaction. These effects are shown in Figure 2, which data is displayed in tabular form in Table 2. See Example 4 for a description of reaction conditions and methodology. Figure 2 shows thermal denaturation profiles for a perfectly base-paired 24-mer ODN duplex and a 24-mer ODN duplex having a single base-pair substitution. These profiles were obtained either in the presence of the exemplary APCs 2-piperidine ethanol (2-PE), l-methyl-2-piperidine methanol (MPM), 1-piperidine ethanol (1-PE), and l-amino-2,6-dimethyl piperidine (AdmP) or in the presence of hybridization buffers commonly employed for hybridization reactions, i.e., PCR Buffer, SSC, and guanidinium isothiocynate (GuSCN).

Importantly, in the presence of an APC, the difference in temperature range over which an oligonucleotide melts (i.e., the ΔHCT) increases to a range of 4- 6°C as compared to a range of 1-2°C under standard conditions. The increase in ΔHCT is a likely consequence of a decrease in melting cooperativity. These data also show that addition of an APC into an ODN hybridization reaction results in an overall decrease in the discrimination between a perfectly base-paired ODN duplex and an ODN duplex having a single base-pair mismatch as evidenced by a ΔTa of 2°C with added APC versus a ΔT_d in the range of 7-8°C under standard hybridization conditions as represented herein by SSC.

It will be understood that, as used herein, the term APC, when used in the context of its effects on perfectly base-paired ODN duplexes versus ODN duplexes having one or more base-pair mismatches, refers to any chemical or any mixture of a chemical in an aqueous or organic environment with buffers, chelators, salts and/or detergents that increases ΔHCT and decreases ΔT_d when referenced to a standard salt solution (e.g., 0.165 M NaCl, 0.01 M Tris pH 7.2, 5 mM EDTA and 0.1% SDS.

APCs of the present invention include, for example, aminoalcohols. As used herein, the term aminoalcohol refers to compounds having both amine and hydroxyl functionality. Compounds with amine functionality have a nitrogen atom, while compounds with hydroxyl functionality have an OH group. The amine group may be a primary, secondary, tertiary, or quaternary amine. In one embodiment, the amine group is a primary amine, while in another embodiment the amine group is a secondary amine, while in another embodiment the amine group is a tertiary amine. The nitrogen atom is not adjacent to a carbonyl group, that is, amides do not contain amines according to the present invention. The hydroxyl group may be a primary, secondary, or tertiary hydroxyl group.

The aminoalcohol may have one, or more than one, e.g., two, three, four, five, etc. amine groups, and may independently have one, or more than one, e.g., two, three, four, five, etc. hydroxyl groups. In one embodiment, the aminoalcohol has a single amine group and a single hydroxyl group. In another embodiment, the aminoalcohol has a single amine group and two hydroxyl groups. In another embodiment, the aminoalcohol has a single amine group and three hydroxyl groups. In another embodiment, the aminoalcohol has a single hydroxyl group and two amine groups. In another embodiment, the aminoalcohol has a single hydroxyl group and three amine groups. In another embodiment, the aminoalcohol has two amine groups and two hydroxyl groups.

The aminoalcohol is preferably soluble in water at some pH. Acids and/or buffers may be added to a mixture of water and aminoalcohol in order to obtain a solution. The addition of acid and/or buffer may convert some or all of the aminoalcohol into a salt form. As used herein, the term aminoalcohol is meant to include both the salt and "non-salt" forms of an aminoalcohol. The pH at which is the aminoalcohol is water-soluble is preferably between 4-10, more preferably between 5-9, and still more preferably between 6-8. Accordingly, the amount of hydrocarbon to which the amine and hydroxyl groups are attached, should preferably be less than that amount which renders the aminoalcohol insoluble in an aqueous composition.

Thus, as indicated above, the term aminoalcohol includes the salt forms of aminoalcohols. As used herein, the salt form of an aminoalcohol refers to the reaction product of the aminoalcohol with an acid, and particularly with a Lewis acid (i.e., a proton donor). The amine group (i.e., the nitrogen atom) of many of the aminoalcohols of the present invention is basic, and can react with an acid to form a salt.

Acids that react with an aminoalcohol, to form an aminoalcohol salt, (where aminoalcohol salts are themselves aminoalcohols of the present invention), include both organic and inorganic acids. The acid may be an organic acid, i.e., be a carboxylic acid of the formula R-COOH where R comprises at least one of H and a carbon-containing organic moiety. The organic moiety may be a hydrocarbon group. The hydrocarbon group may have 1 to 22 carbon atoms, i.e., R may be Cι-C . The hydrocarbon group may be saturated, or it may be unsaturated. The hydrocarbon group may be cyclic, or it may be acyclic. In one embodiment, the hydrocarbon group is Ci-Cio, while in another embodiment the hydrocarbon group is a relatively short-chain organic group, i.e., R is a Cι-C₅ group. Organic acids with hydrocarbon R groups include, without limitation, acetic acid, benzoic acid, butyric acid, cyclohexanecarboxylic acid, decanoic acid, 2- ethylbutyic acid, 2-ethylhexanoic acid, heptanoic acid, hexanoic acid, lauric acid, myristic acid, nonanoic acid, octanoic acid, palmitic acid, and propionic acid.

The R group of the organic acid may be a halocarbon. A halocarbon according to the present invention is a hydrocarbon as described above having one or more of the hydrogens replaced by halogen(s). As used herein, bromide, chloride, fluoride and iodide are halogens, where bromide, chloride and fluoride are preferred halogens. Fluoride and chloride, being less reactive halogens when bonded to carbon, are preferred halogens.

Exemplary halogenated carboxylic acids include, without limitation, chloroacetic acid, dichloroacetic acid, dichlorofluoroacetic acid, difluoroacetic acid, chlorofluoroacetic acid, fluoroacetic acid, trichloroacetic acid, trifluoroacetic acid, etc.

Inorganic acids may likewise be used to prepare the salt of the aminoalcohol. Suitable inorganic acids include, without limitation, hydrochloric acid, phosphoric acid, nitric acid, and hydrobromic acid.

The aminoalcohol may contain functionality in addition to an amine group and a hydroxyl group. For instance, the aminoalcohol may contain ether functionality.

The nitrogen atom of the amine group may form part of a heterocyclic ring. Exemplary heterocyclic rings that contain a nitrogen atom, and that may be substituted either directly or indirectly with one or more hydroxyl groups, include aziridine, azetidine, azolidine (pyrrolidine), piperidine (perhydroazine), pyrrole, imidazole, pyridine, pyrimidine, purine, indole, quinoline, isoquinoline, pyrazine, perhydroquinoline, and perhydroisoquinoline. As used herein, indirect substitution of a heterocyclic ring with a hydroxyl group means that there are some atoms, e.g., a hydrocarbyl group, disposed between the hydroxyl group and the heterocyclic ring. Suitable aminoalcohols are piperidine derivatives, i.e., compounds having at least one piperidine nucleus and at least one hydroxyl group. A hydroxyl group may be joined directed to a piperidine ring, as in 4-hydroxypiperidine, or a hydrocarbon group may be disposed between the piperidine group and the hydroxyl group, as in 2-piperidineethanol. The nitrogen atom of the piperidine ring may be unsubstituted, i.e., be bonded to a hydrogen atom as in 4-hydroxypiperidine, or may be substituted, e.g., be bonded to a non-hydrogen atom as in l-ethyl-4-hydroxypiperidine. Suitable aminoalcohols that are piperidine derivatives include, without limitation, 4- hydroxypiperidine, l-methyl-3-piperidinemethanol, 4,4'-trimethylenebis(l - piperidineethanol), 3-piperidinemethanol, l-ethyl-4-hydroxypiperidine, 2- piperidineethanol, 3-hydroxy-l-methylpiperidine, l-ethyl-3-hydoxypiperidine, 4- hydroxy- 1 -methylpiperidine, 1 -methyl-2-piperidinemethanol, 2-piperidinemethanol, and 2,2,6,6-tetramethyl-4-piperidinol.

Piperazine derivatives are another suitable aminoalcohol of the present invention. Piperazine derivatives that are also aminoalcohols are compounds having at least one piperazine nucleus and at least one hydroxyl group. A hydroxyl group may be joined directed to a piperazine ring, or a hydrocarbon group may be disposed between the piperazine group and the hydroxyl group so that the hydroxyl group is indirectly joined to the piperazine ring. The hydrocarbon group may join the piperazine group at any ring atom. A nitrogen atom of the piperazine ring may be unsubstituted, i.e., be bonded to a hydrogen atom, or may be substituted, i.e., be bonded to a non-hydrogen atom. Suitable aminoalcohols that are piperazine derivatives include, without limitation, l,4-bis(2-hydroxyethyl)piperazine and l-(2-hydroxyethyl)piperazine.

Aminoalcohols, including salt forms thereof, are commercially available from many commercial supply houses including, without limitation, Aldrich (Milwaukee, WI; www.aldrich.sial.com); EM Industries, Inc. (Hawthorne, NY http://www.emscience.com); Lancaster Synthesis, Inc. (Windham, NH http://www.lancaster.co.uk); Spectrum Quality Product, Inc. (New Brunswick, NJ http://www.spectrumchemical.com); and Stepan Company (Northfield, IL http://www.stepan.com). The salt form of an aminoalcohol may be purchased from a commercial supply house, some of which are listed above, or may be synthesized. Salts are readily synthesized by mixing the aminoalcohol with an acid. Upon being mixed, the aminoalcohol and acid will spontaneously form a salt. Typically, the salt-forming reaction is performed in a suitable solvent, such as water. After formation of the salt, the solvent may be removed, e.g., by evaporation, or the salt may precipitate and then be recovered by filtration. Alternatively, the solution of salt may be used in the present invention without isolation.

The salt need not be formed prior to one or both of the aminoalcohol or acid being contacted with nucleic acid. Thus, a solution of nucleic acid and aminoalcohol may be treated with an acid, to form a mixture of nucleic acid, and one or more of aminoalcohol, acid, and salt of aminoalcohol and acid. Alternatively, a solution of nucleic acid and acid may be treated with aminoalcohol, to form the same composition. Typically, if the aminoalcohol is added to an aqueous composition having a pH of less than 7, i.e., an acidic solution, at least some of the aminoalcohol will form a salt with at least some ofthe acid responsible for the solution having a pH of less than 7.

Compositions

As noted above, the present invention provides compositions comprising one or more APCs, either as specified above or as identified through readily available techniques described in detail below. Also provided are methods employing these inventive compositions, which methods rely on a reduction in the specificity of oligonucleotide hybridization reactions that results from addition of an APC.

The present compositions may further comprise an oligonucleotide. When included in such a composition, the oligonucleotide is preferably at a concentration of from about 10^" to 10^" g/ml.

In addition to the oligonucleotide and APC, other components may be present in compositions of the present invention. As noted herein, an APC is useful within the context of the present invention if it dissolves or is miscible at a concentration of from about 1.0 mM to about 10 M in water, other protic, or aprotic solvent. Other suitable optional components include, without limitation, at least one of a buffer, detergent and chelator.

In some embodiments, the compositions may also contain one or more enzyme, such as polymerases and/or ligases. A polymerase is desirable in compositions that are used in amplification or primer extension reactions including, but not limited to, the polymerase chain reaction (PCR), in vitro transcription and translation reactions, nucleic acid sequencing and the like. One of ordinary skill in the art will recognize that the selection of an appropriate polymerase or ligase will depend on the precise application employed. Thus, for example, in a typical polymerase chain reaction, it may be desirable to use one of the commercially available thermal stable DNA polymerases, such as Taq DNA polymerase (Perkin Elmer, Norwalk, CT). For guidance in the selection of enzymes, see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratories, 2^nd ed., 1989. Preferably, the APC does not inactivate the enzyme, where enzyme activity in an APC solution may be measured according to the use of the enzyme and/or in accordance with standard conditions provided by the supplier. For example, in amplification reactions, duplicate reactions with and without added APC may be run. An APC does not inactivate an enzyme if 10% of its activity, as determined in standard reactions conditions for that enzyme, is retained.

Identification of APCs within the Scope ofthe Invention

Additional APCs within the scope of the present invention may be readily identified out of a panel of test compounds by determining the effect of the test compounds on the thermal denaturation parameters discussed, supra, by methods disclosed herein or as otherwise commonly employed in the art. Suitable methodology for the generation of thermal denaturation profiles include, but are not limited to, use of nylon bead supports (ODN-Bead), nylon tip supports (ODN-Tips) and nylon membranes. Each of the methods described herein have in common the immobilization, via attachment, of a target oligonucleotide, i.e., the capture oligonucleotide, on an insoluble support. While each of these methods is described in considerable detail herein, it will be recognized that alternative methods for generating thermal denaturation parameters may be equally suited for the identification and characterization of putative APCs. Thus, the present invention is not limited by the presently disclosed methodology.

Thermal denaturation parameters may be derived using the oligonucleotide (ODN) bead technology described in Van Ness et al, Nucl. Acids Res. 19:3345, 1991. T_d and T_opt values may be determined using the ODN-beads in various hybridization solutions containing putative or actual APCs. A capture oligonucleotide is immobilized on the nylon bead support and unattached amine-modified ODNs are labeled by reaction with amine-reactive fluorochromes. See, e.g., Example 1. The derived ODN preparation is divided into 3 portions and each portion is reacted with (a) 20-fold molar excess of Texas Red sulfonyl chloride (Molecular Probes, Eugene, OR); (b) 20-fold molar excess of Lissamine sulfonyl chloride (Molecular Probes, Eugene, OR); or (c) 20-fold molar excess of fluorescein isothiocyanate. The final reaction conditions consist of 0.15 M sodium borate at pH 8.3 for 1 hour at room temperature. The unreacted fluorochromes are removed by size exclusion chromatography on a G- 50 Sephadex column.

For the determination of ODN/ODN T_d from the ODN-bead, fluorescently-labeled ODN is incubated in various hybridization solutions with a complementary capture ODN immobilized on ODN-beads. 3/32nd inch diameter beads are prepared as described in Van Ness, Id., and contain 0.01 to 1.2 mg ODN/bead. From 5 to 5000 ng of ODN are hybridized in 300-400 μl volumes at various temperatures (19-25°C) for 5-30 minutes with constant agitation. The beads are washed with 3 x 1 ml of the respective hybridization solution, and then once with the respective melting solution at the starting temperature of the melting process. The beads in 300- 400 μl of the respective melting solution are then placed in a 0-15°C water bath. At 5 minute intervals, the temperature is raised 5°C, the solution transferred into a well of a microtiter plate, and fresh solution (5°C below the next increment) is added to the beads. The "melting" or duplex dissociation is conducted over a temperature range of 15°C to 95°C. Fluorescence is measured with a commercial fluorescence plate reader, such as is available from Wallac (Turku, Findland).

To calculate the T_d, cumulative counts eluted at each temperature are plotted against temperature. The temperature at which 50% of the material is dissociated from the bead is the T_d. ΔT is defined as the difference in T_d obtained for an ODN/ODN duplex in a control hybridization solution such as IX SSC and a test hybridization solution containing a putative APC. HCT is the difference between the temperature at which 20% of the labeled ODN is dissociated and at which 80% of the labeled ODN is dissociated. The HCT of an ODN duplex can be measured essentially as described by Martinson for the thermal elution of DNA or RNA duplexes or hybrids from hydroxylapatite. Biochemistry 2:145-165, 1973. ΔHCT is the difference in the HCT for the control solution and the test compound. As discussed, supra, an APC is defined as a compound that decreases ΔT_d and increases HCT thus resulting in a positive ΔHCT (i.e., positive HCT_Apc - HCTssc)- Thermal denaturation parameters for RNA/ODN or DNA ODN may alternatively be determined with ODN immobilized on nylon membranes such as those available from Schleicher & Schuell, Keene, N.H. ³²P-labeled ODN (e.g., 3 '-end labeled with terminal transferase; see Sambrook et al, supra) is incubated with 0.5 cm² pieces of membrane, in the desired hybridization solutions. For the non-covalent immobilization of genomic DNA onto nylon membranes, purified DNA is denatured in 0.3 M NaOH at 20°C for 10 minutes. An equal volume of 2 M ammonium acetate is added and the sample applied to NYTRAN membranes assembled in a slot blot apparatus. RNA is denatured in 4.6 M formaldehyde-6X SSC (0.9 M NaCl, 90 mM sodium citrate) for 15 min. at 60°C and applied to the membranes as above. After immobilization of the nucleic acids, the filters are baked at 80°C for 2 hours, then stored dry at ambient temperature. The hybridization and dissociation reactions are then performed as described above for the nylon bead solid supports.

To determine the T_optODN (the temperature at which the maximum rate of hybridization of target nucleic acid to ODNs occurs, under near stringent to stringent conditions; i.e., -20 to -5°C below the Ta), complementary P-labeled ODN is hybridized to either covalently immobilized ODN sequences on the ODN-bead as described above, or in a sandwich assay format when RNA is used as the target nucleic acid. The hybridizations are performed over a 40°C range (± 20°C around the T_d of the respective duplex in 5°C increments). The extent of hybridization is then measured as a function of temperature of the respective hybridization.

Thermal transitions may also be determined in solution (T_m) by recording at 260 nm using a Gilford System 2600 UV-VIS spectrophotometer equipped with a Gilford 2527 Thermo-programmer. ODNs (2 mM/strand) are dissolved in the respective hybridization or melting solutions. The ODN mixtures are heated to 85°C, then cooled to 10-15°C to allow hybridization. The samples are slowly heated to 85°C employing a temperature increase of 0.5°C/min. Absorbance versus time is recorded, and the first derivative is computed automatically. The T_m values are determined using the first derivative maxima.

Thermal denaturation parameters may preferably be obtained by the ODN-Tip methodology described in detail in Example 1, infra. Briefly, a capture oligonucleotide is attached to nylon tips by, e.g., using the approach described by Van Ness et al. Nucl. Acids Res. 79:3345, 1991. The nylon tips are then contacted with a solution containing a cognate oligonucleotide. After hybridization, tips are washed free of unhybridized ODN. The tips are next passaged stepwise through a series of wells on, e.g., a 96-well PCR plate. The temperature is increased incrementally with each subsequent step. The amount of single-strand ODN is measured on a commercially available fluorescence plate reader and the thermodynamic parameters calculated.

It will be apparent to one of ordinary skill in the art that alternative methods readily available in the art may also be suitable for the measurement of ODN/ODN thermal denaturation parameters. The present invention is not, therefore, limited in scope by the methodologies described in the instant disclosure.

Methods Employing APC Based Compositions

Compositions of the present invention may be used in a variety of methods where it is advantageous to reduce the specificity between a perfectly base- paired oligonucleotide duplex and an oligonucleotide duplex containing one or more base-pair mismatches. Thus, methods contemplated as within the scope of the present invention include those that require, or are improved by, a reduction in ΔT_d (such as methods wherein ΔT_d = 0) and stringency and/or an increase in HCT. Such applications include, but are not limited to, enzymatic reactions commonly used in molecular biology that employ one or more enzymes such as polymerases and or ligases. Such enzymatic reactions include, for example, in vitro transcription and translation, reverse transcription, nucleic acid sequencing, the polymerase chain reaction, nucleic acid ligations and the like. In addition to enzyme based applications, the inventive compositions will find utility in a variety of non-enzyme based methodologies that rely on nucleic acid hybridization and that are benefited by reduced hybrid specificity. Exemplary applications include the various conventional molecular biology technologies utilizing oligonucleotide hybridization such as Southern and Northern hybridization, differential hybridization, spot or slot blotting, colony hybridization, and screening of cDNA libraries. See, e.g., Sambrook, et al., supra. In addition, compositions of the present invention will also find utility in a variety of cytogenetic techniques where it is desired that the oligonucleotide hybridization specificity be reduced. These techniques include comparative genomic hybridization, spectral karyotyping, in situ hybridization (e.g., RISH and FISH), restriction-landmark genomic scanning, representational difference analysis, differential display, serial analysis of gene expression and microarray techniques.

Representative enzyme-based and non-enzyme-based applications are provided below. It will be apparent to one of ordinary skill in the art that there may be alternative applications for the inventive APCs, other than those specifically recited, where it is advantageous to reduce the hybridization specificity between one oligonucleotide and a corresponding target oligonucleotide. Thus, the present invention is not limited in scope by the specific applications discussed herein.

The polymerase chain reaction (PCR), a method for amplifying specific nucleic acid sequences, permits the rapid detection of nucleic acids present in a sample in what was previously an undetectably low quantity. See, e.g., U.S. Patent Nos. 4,683,195; 4,683,202; and 4,965,188. There is a need in the art for methodologies that permit the PCR amplification of nucleic acid fragments where the primer sequences base-pair imperfectly with the cognate sequence on the target nucleic acid template. For example, it is well established that viruses as well as live viral-based vaccines incorporate multiple nucleotide substitutions or deletions over the course of evolution or passaging, respectively. See Behr, M.A., et al., Science 284:1520-1523, 1999 (discussing accumulation of mutations in tuberculosis vaccines that result from passaging live viral vaccines under attenuating conditions). Thus, it is advantageous that existing primer resources be able to be utilized to amplify sequences from such sources notwithstanding minor or substantial changes in target nucleic acid sequence.

By way of example, and not limitation, it is well known that the Human Immunodeficiency Virus Type I (i.e., HIV-1) has undergone and continues to undergo significant changes in nucleotide sequence over the course of time. Reviewed in U.S. Patent No. 5,599,662. Thus, under conventional PCR amplification technologies, ODN primers must be redesigned periodically to achieve reliable amplification of HIV-1 gene segments from various viral isolates where the individual viral isolates differ as to nucleic acid sequence. See, e.g., Kwok, Ann. Med. 24:211-214, 1992; Coutlee, Mol. Cell. Probes 5:241-259, 1991; and U.S. Patent Nos. 5,008,182 and 5,176,775, (describing PCR and probe hybridization methodologies for amplification and detection of HIV-1 nucleic acid). In the absence of knowledge as to a given isolate's nucleic acid sequence, it has heretofore been impossible to design, with certainty, amplification primers that will precisely base-pair and thus hybridize to the new viral isolate. In order for one of ordinary skill in the art to continue to utilize existing amplification primers, the hybridization specificity of existing oligonucleotides must be reduced such that ODN duplexes may be formed even when there is imperfect base-pairing.

The APCs of the present invention will, therefore, be useful in permitting the hybridization to and amplification of viral template sequences that vary in nucleotide sequence from the cognate amplification primers. Thus, APCs may, for example, be added to conventional PCR reactions in concentrations that reduce, and preferably minimize, the discrimination between a perfectly base-paired oligonucleotide duplex and an oligonucleotide duplex having one or more base-pair mismatch.

The concentration of an APC to be added to a given PCR reaction will be an amount less than that which destroys polymerase activity. Such a concentration may be determined by, for example, titrating a test PCR reaction with increasing amounts of a given APC. APCs of the present invention may be effective in the range of from about 1 mM to 10 M. More preferred concentrations of APC are from 5 mM to 5M. Still more preferred are APCs in the concentration range of 10 mM to 1M. Still more preferred are APCs in the range of 100 mM to 1M. Thus, it will be apparent to one of ordinary skill in the art that, in a test PCR reaction, increasing amounts of an APC within the range of 1 mM to 10 M may be tested for its effect on polymerase activity. By the present invention, any concentration may be employed as long as it does not reduce polymerase activity by an excess of 90% as compared to a control reaction using conventional reaction conditions recommended by the supplier of the given polymerase.

In addition to APCs, polymerase chain reactions will additionally contain a first (e.g., forward) and second (e.g., reverse) oligonucleotide; target oligonucleotide; dNTPs (i.e., dATP, dTTP, dCTP and dGTP); a thermostable polymerase such as Taq DNA polymerase; and a suitable amplification buffer. One of ordinary skill in the art will recognize that the precise reaction conditions may vary in accordance with the specific application employed. Melting, annealing and polymerization temperatures will be optimized as required for the target and primer oligonucleotide combination utilized according to standard methods readily available in the art. See, e.g., Wu, W. et al., in Methods in Gene Biotechnology, 1997. More than one APC may be used simultaneously to further reduce hybridization specificity as required for a given application. As well, alternative or additional components may be added to improve the hybridization properties such as reduction in ΔT_d or increase in ΔHCT. Alternative components may also be added to enhance the polymerization reaction. Each of these modifications to standard reaction conditions may be performed without undue experimentation by one of ordinary skill in the art.

APCs may also be invaluable in random-priming applications where it is advantageous to reduce or minimize ODN/ODN hybridization specificity. One exemplary application is the use of APCs in priming reactions with degenerate oligonucleotides in in vitro methods for generating pools of mutagenized nucleic acid fragments. See, e.g., Shao, Z. et al, Nucl. Acids. Res. 26(2):68l-6%3, 1998 (discussing the use of random primers as a tool for directed evolution). More specifically, Shao et al., discuss the use of random-primers and recombination to amplify specific nucleic acid sequences in order to generate a pool of mutagenized DNA fragments. These DNA fragments may be reincorporated within the sequence context of a wild-type gene to serve as a pool of randomly mutated genes from which new proteins may be isolated that have such desired biological properties as, for example, increased enzyme activity or stability as well as decreased in vivo toxicity. By applying APCs of the present invention to such a methodology, it may be possible to utilize the random-priming oligonucleotide sets to amplify similar DNA fragments from a variety of genetic isoforms that would not otherwise be amplified with the primers. Similarly, APCs may be used to amplify DNA fragments from gene homologues from a variety of unrelated species that would not be amplified using random-primers designed for the original species. Thus, APCs provide the opportunity for amplification of DNA fragments without the necessity of first determining the nucleic acid sequence of the target nucleic acid and without having to design a new set of oligonucleotide primers for every new gene of interest.

As noted above, APCs of the present invention will also find utility in a variety of detection methods that do not employ polymerase-based or other enzymatic reactions. These methods include, but are not limited to, those molecular biology and cytogenetic applications listed herein, supra. Thus, APCs may, for example, be used in a variety of cytogenetic and molecular genetic techniques for the genome-wide screening of alterations in copy number, structure and expression of genes and DNA sequences such as comparative genomic hybridization, in situ hybridization, as well as in a variety of array and microarray techniques.

Comparative genomic hybridization (CGH) is a cytogenetic technique that allows the screening for DNA sequence copy-number changes and provides a map of chromosomal regions that are gained or lost in a DNA sample. Reviewed recently by Forozan, F. et al, Trends in Genet. 13(10):4Q5-409, 1997. For a description of the CGH methodology, see also Kallioniemi, O-P et al., Genes Chromosomes Cancer 70:231-243, 1994 and Kallioniemi, A. et al, in Laboratory Methods for the Detection of Mutations and Polymorphisms, pp. 273-285 (Taylor, G.R., ed., 1997). The CGH technology is used extensively in cancer research to investigate alterations in DNA copy number related to pathogenesis. CGH is also used to diagnose unbalanced chromosomal rearrangements and in determining the origin of extrachromosomal material. See, e.g., Bryndorf, T. et al, Am. J. Hum. Genet. 57:1211- 1220, 1995; Levy, B. et al, Cytogenet. Cell Genet. 76:68-71, 1997; Wegner, R.D. et al, Prenat. Diag. 16:741-748, 1996; and Wang, B.B. et al., Prenat. Diag. 75:1115-1119, 1995.

CGH is based on the in situ hybridization technology and utilizes differentially labeled test and reference DNAs that are co-hybridized to normal metaphase chromosomal spreads. Because the test and reference DNAs are labeled with distinct fluorochromes, differences in DNA copy number are detectable by measuring the testireference fluorescence ratio obtained by means of a digital image analysis system. Thus, gene duplications are seen as an increase in test DNA fluorescence relative to reference DNA fluorescence while gene deletions are indicated by the decrease in this ratio. Considerable resources have been committed to the development of panels of CGH test and reference DNAs. Because of the high degree of sequence identity required between the test and reference DNAs and their respective cognate target sequences, these DNAs may not hybridize to target sequences having one or more base-pair mismatches. These base-pair mismatches may occur either through mutation ofthe target sequence or where the DNA panels are to be used for detection of homologous sequences present in the chromosomes of species other than the one for which the panel DNAs were originally designed.

For example, CGH has recently been expanded to the analysis of mouse cells, which will make it possible to apply this technique for tracing the genetic basis of tumor development and progression in transgenic mouse models of human cancer. Donehower, L.A. et al, Prog. Clin. Biol. Res. 395:1-11, 1996. Because APCs of the present invention allow a reduction of ODN/ODN hybridization specificity, these compounds may permit the direct comparison of, e.g., human genomic sequences implicated in neoplastic disease with homologous regions in the transgenic mouse model. Thus, compositions and methods of the present invention may be employed by including APCs in CGH reactions to permit a reduction in hybridization specificity between test or reference DNAs and their coπesponding target sequences.

In related applications, APCs may be used in CGH methodologies to classify chromosomal regions on the basis of species to species relatedness. With the existing CGH technology, cross species homologues may only be identified when there is a high degree of sequence identity. By applying APC compositions of the present invention, it may be possible to expand CGH utility by reducing oligonucleotide hybridization specificity thereby permitting the detection of sequences that have significantly lower degrees of sequence identity. APCs may similarly be utilized in microarray-based methods where reduced hybridization specificity is desired. The term "microarray" includes an array of linear or two-dimensional regions, each region being discrete from the other and each region having a finite area, formed on the surface of a solid support. Methods for fabricating microarrays are readily available in the art and have been described, e.g., in Brown et al. U.S. Patent No. 5,807,522; Wang et al, U.S. Patent No. 5,922,617; Billings et al., FASEB 5:28-34, 1991; Chee et al, Science 274:610-614, 1996; Drmanac et al., Electrophoreses, 73:566-573, 1992.

Microarray-based methods have been employed, for example, in the identification of putative human and environmental toxicants and their potential modes of action, Medlin, J.F., Environ Health Perspect. 107 (5): A156-8, 1999; Nuwaysir, E.F. et al, Mol. Carcinog. 24(3):\53-\59, 1999; and Schena, M. et al, Trends Biotechnol i<5(7,):301-306, 1998; for assessing the complexity of radiation stress responses, Fornace A.J. et al, Gene Expr. 7 (4-6) :387 -400, 1999 and Amundson, S.A. et al. Oncogene 18(24):3666-3672, 1999; to identify changes in gene expression levels in neoplastic diseases, Bennicelli, J.L. et al, Curr. Opin. Oncol. ll(4):267-74, 1999; and to study the evolution of tuberculosis vaccines, Behr, M.A. et al, Science 284(5419):\520-\523, 1999.

By providing a systematic approach to hybridization-based detection, microarray-based methods allow the monitoring of expression levels of hundreds or thousands of genes simultaneously. For example, vast numbers of genes that undergo upregulated expression levels in response to challenge with known toxicants have been identified and their corresponding cDNAs have been isolated and sequenced. Based on these nucleic acid sequences, panels of synthetic oligonucleotides have been produced such that each toxin upregulated gene is represented by a coπesponding oligonucleotide. Individual oligonucleotides may be attached covalently, for example by the oligonucleotide' s 5' end, to a single position of a glass slide; 96-well plate; nylon beads, tips, or membranes; or other suitable solid support. By similarly attaching each member of the oligonucleotide panel to a position on the solid support, an array is created that corresponds to all genes that are known to be upregulated by toxic compounds.

Test substances may be administered in vivo to an animal or ex vivo to cells in culture and cDNA's synthesized from animal tissue or culture cell mRNAs. These cDNA's may be labeled, for example, with a fluorochrome, a radionucleotide, or a cleavable tag (see, e.g., PCT International Publication Nos. WO 97/27331; WO 97/27325; WO 97/27327; and WO 99/05319), and applied to the oligonucleotide array. By measuring the resulting signal at each position in the array, the relative toxicity of the test substance may be ascertained and the specific genes that are upregulated may be identified.

The microarray-based methods presently available in the art are limited in utility, however, because the synthetic oligonucleotide panels that are used to create the microarray are highly specific to only those cDNAs that have been isolated and sequenced. Because of this high degree in ODN/ODN specificity, genetic isoforms or homologous genes from other species may not be detected by the existing microarray- based techniques. APCs of the present invention provide the opportunity to greatly expand the utility of existing microarray-based methods by broadening the possible applications for existing oligonucleotide panels. Addition of APCs to any of the microarray-based applications discussed in the above references will enhance the use of existing ODN panels by permitting reducing ODN/ODN hybridization specificity. Each of the above utilities for APCs in conventional molecular biology applications as well as in various cytogenetic and molecular genetic applications as discussed above are merely exemplary. It will be apparent that the presently disclosed APCs may find a wide range of alternative uses where it is advantageous to reduce the hybridization specificity of an ODN/ODN duplex. These uses are fully within the spirit and scope of the present invention which is not limited to the specific utilities provided herein.

EXAMPLES

The following examples are provided by means of illustration, not limitation. In the following Examples and Tables, T_d, ΔT_d, HCT, and ΔHCT are reported in °C.

EXAMPLE 1

MATERIALS

Solutions and Reagents

As used herein, filter wash (FW) is 0.09 M NaCl, 540 mM Tris pH 7.6, 25 mM EDTA. FW with 0.1% sodium dodecyl sulfate (SDS) is SDS/FW.

Hybridization solutions contain the specified concentration of APC (purchased from

Aldrich, Milwaukee, WI), 50 mM Tris pH 7.6 and 25 mM EDTA. Formamide hybridization solution contains 30% formamide, 0.09 M NaCl, 40 mM Tris-HCl pH 7.6, 5 mM EDTA and 0.1% SDS. GuSCN was purchased from GIBCO BRL Life Technologies (Gaithersburg, MD). SSC, NaCl, Tris, EDTA and SDS were purchased from Sigma (St. Louis, MO). RapidHybe was purchased from Amersham (Piscatoway, NJ).

Oligonucleotides

Oligonucleotides were synthesized on a commercial synthesizer using standard cyanoethyl-N,N-diisopropylamino-phosphoramidite (CED-phosphoramidite) chemistry. Amine tails were incorporated onto the 5 '-end using the commercially available N-monomethoxytritylaminohex-6-yloxy-CED-phosphoramidite. As an alternative, oligonucleotides may be commercially purchased (Midland Certified Reagents, Midland, TX).

Probe oligonucleotides were prepared by reacting amine-modified oligonucleotides with amine-reactive fluorochromes. The derived oligonucleotide preparation was divided into three portions and each portion was reacted with either (a) a 20-fold molar excess of Texas Red sulfonyl chloride (Molecular Probes, Eugene, OR), (b) a 20-fold molar excess of Lissamine sulfonyl chloride (Molecular Probes, Eugene, OR), or (c) a 20-fold molar excess of fluorescein isothiocyanate. The final reaction conditions consisted of 0.15 M sodium borate (pH 8.3) for one hour at room temperature. The unreacted fluorochromes were removed by size exclusion chromatography on a G-50 Sephadex column.

EXAMPLE 2 DETERMINATION OF THERMAL DENATURATION PARAMETERS

This example describes the preparation and use of oligonucleotide nylon tip supports (ODN-Tips) for determining thermal denaturation parameters for oligonucleotide duplexes melted in the presence of APCs ofthe present invention. A high throughput method for the measurement of the thermodynamic properties of oligonucleotide duplexes was developed. The method allows thousands of solution samples to be scanned for their ability to modulate the thermodynamic parameters of the helical to coil transition (HCT) of oligonucleotide duplexes. This method employs a solid support designed to fit in a Cetus plate (or the well of a plate designed for 96 well PCR format) and requires about 40 μl of volume to be completely covered by liquid.

One member of the oligonucleotide duplex, i.e., the "capture" oligonucleotide, is immobilized on each nylon tip as described by Van Ness et al, Nucleic Acids Res. 79:3345-3350, 1991. A hybridization step is then performed to allow the oligonucleotide duplexes to form on each tip. The hybridization step can be performed en masse in a single container or individually in the wells of a PCR plate. It is therefore possible for every tip of a 96 member array of tips to possess a different oligonucleotide duplex. After the hybridization step, the tips are washed of unhybridized, single- stranded oligonucleotide and then placed in a PCR plate mounted on a thermocycler. In the case of a 1x8 or 1x12 format, the tips are then moved stepwise through a series of wells. Typically, the temperature in each subsequent set of wells is increased in increments of 5°C and maintained for 1 to 5 minutes. For example, tips in a 1x12 format are initially placed in a row at 10°C. The thermocycler is then programmed to ramp through 16 steps at 2 minute intervals with 5°C increments of temperature. The tip array is moved from row to row 15 seconds prior to the temperature increase. In this format, 12 solutions can be studied using two plates of solution. In a 96 tip format, entire plates of solution are moved off and on the thermocycler at the timed interval. Fluorescent probes are commonly used in this format and have minimal effect on the measured Ta values described herein. The use of radiolabeled or fluorescent probes permit a wide variety of solutions to be measured since there is no requirement of optical clarity, in contrast to, e.g., melt curves derived by UV spectrometry (hyperchromicity shifts). Fluorescence may be measured, for example, with a micro titer plate fluorescence reader. The data are directly imported into a spreadsheet program, such as EXCEL, that calculates the stability, enthalpy, helical to coil transition, and temperature range and graphs the results. Typically, a 1x12 format that measures 12 solutions at once can be completed within one hour, including set up and data reduction. For the determination of ODN/ODN Ta from the oligonucleotide-tip, fluorescently-labeled oligonucleotide is incubated in various hybridization solutions with a complementary capture oligonucleotide immobilized on oligonucleotide-tips. From 5 to 5000 ng of oligonucleotide are hybridized in 100-200 μl volumes at various temperatures (19-26°C) for 10 to 60 minutes. The tips are washed three times with FW/SDS, and then three times with FW at room temperature. The solution plate is then placed on the thermocycler and equilibrated to the starting temperature ofthe melt. The tips are placed in the first test row ofthe melting solution.

At one to five minute intervals, the temperature is raised 5°C, and the tips are moved into a new well of the microtiter plate. The melting, or duplex dissociation, is conducted over a temperature range of 10°C to 95°C. Fluorescence is measured with a commercial fluorescence plate reader.

To calculate the Ta, cumulative relative fluorescent units (RFUs) eluted at each temperature is plotted vs. temperature (°C). The temperature at which 50% of the material is dissociated from the tip is the T_d. The HCT is calculated as the difference between the temperature at which a value of α equals 0.2 for a given oligonucleotide duplex and the temperature at which a value for α equals 0.8 for the same oligonucleotide duplex.

Melting temperature measurements described in the following examples were performed using the tip methodology described in this example. Alternative methods may be employed, where desired, to make similar measurements. For example, other solid supports that may be used include the nylon beads, nylon membranes and related supports as described in the present disclosure, supra, or as otherwise readily available in the art. The use of tips is generally prefeπed in the present applications as it affords a high throughput method for the measurement of the thermodynamic properties of oligonucleotide duplexes. EXAMPLE 3

APC BASED COMPOSITIONS NEUTRALIZE DIFFERENCES

IN G+C CONTENT AND REDUCE ΔT_D

This example shows that APCs of the present invention possess the property of neutralizing differences in G+C and A+T base-pairing strength and of simultaneously lowering discrimination (i.e., reducing ΔT_d).

The following oligonucleotides, each having a different G+C content, were synthesized and used in experiments to determine the effect of inventive APCs on thermal denaturation parameters as a function of G+C content:

Capture 36-mer: 5 '-Hexylamine-GCAGCCTCGCGGAGG-

CGGATGATCGTCATTAGTATT-3 '

27% G+C 5 '-Texas Red-AATACTAATGACGATCAT-3 ' 44% G+C 5 '-Texas Red-ACTAATGACGATCATCCG-3 ' 50% G+C 5 '-Texas Red-AATGACGATCATCCGCCT-3 ' 61% G+C 5 '-Texas Red-GACGATCATCCGCCTCCG-3 ' 83% G+C 5 '-Texas Red-CCGCCTCCGCGAGGCTGC-3 ' The capture 36-mer ODN was attached to each tip as described in Example 2. The tips were contacted with a solution containing one of the 27%, 44%, 50%, 61%, or 83% G+C content 18-mer ODNs in the presence of either the APC 2- piperidine ethanol (2-PE, 0.7 M), IX SSC, 2M GuSCN, or PCR buffer.

Table 1 and Figures 3A-D show that addition of the APC 2-piperidine ethanol neutralized the difference in the discrimination temperature (Ta) between a high G+C content oligonucleotide (83%) and a low G+C content oligonucleotide (27%) as compared to parallel control reactions containing SSC, GuSCN or a standard buffer commonly used for PCR amplification reactions. SSC is commonly used in oligonucleotide hybridization reactions, such as Southern and Northern hybridizations, and GuSCN is frequently used in hybridization reactions to increase duplex specificity. In addition, 2-piperidine ethanol also caused an overall increase in the helical to coil transition (HCT) relative to the corresponding control reactions. "ND" stands for not determined.

Table 1

EXAMPLE 4 APCS INCREASE ΔHCT AND REDUCE ΔT_D

This example compares the thermal denaturation parameters a perfectly base-paired ODN duplex and an ODN duplex containing a single base-pair mismatch.

The following oligonucleotides were used to measure the difference in

T_d between a wild type (WT) 24-mer ODN and a mutant (MT) 24-mer ODN. The wild type ODN forms a perfectly base-paired duplex and the mutant ODN forms a duplex having a single base-pair mismatches in the middle ofthe duplex. The capture ODN for this example is the "capture 30-mer."

Capture 30-mer:

5 '-hexylamine-GTCATACTCCTGCTTGCTGATCCACATCTG-3 ' WT 24-mer: 5 '-Texas Red-TGTGGATCAGGAAGCAGGAGTATG-3 ' MT 24-mer: 5 '-Texas Red- TGTGGATCAG(dN)AAGCAGGAGTATG-3 ' Table 2 and Figures 2A-G display the thermal denaturation parameters obtained in hybridization reactions containing either 1M 2-piperidine ethanol (2-PR); 1M 1 -methyl-2-piperidine methanol (MPM); 1M 1 -piperidine ethanol (1-PE); 1M 1- amino-2,6-dimethyl piperidine (AdmP); IX SSC; 2M guanidinium isothiocyanate (GuSCN); or PCR buffer.

Table 2

These data show that APCs according to the present invention promoted the non-specific hybridization of oligonucleotide duplexes, as evidenced by the increase in ΔHCT and the corresponding decrease in ΔT_d, as compared to the respective thermodynamic parameters obtained in the control solutions.

EXAMPLE 5

EFFECT OF APC CONCENTRATION DEPENDENCE ON ODN/ODN

THERMAL DENATURATION PARAMETERS

This example discloses the effect of APC concentration on HCT and T_d. The data displayed in Table 3 were obtained with the oligonucleotides presented in Example 4 using increasing concentrations of the APC piperidine methanol (PM). These data show that the reduction in discrimination, i.e., a lowering of ΔTd, between a perfectly base-paired ODN duplex and an ODN duplex containing a single base-pair mismatch did not depend upon the concentration of APC within the range tested (0.5- 2.5 M PM). Similarly, the helical to coil transition (HCT) temperature was also unaffected by PM concentration. In fact, no change in HCT was detected at up to 6 M PM. Further, these data show that the discrimination temperature (T_d) increased as a linear function of PM concentration.

Table 3

EXAMPLE 6 EFFECT OF Low CONCENTRATIONS OF APC ON HELICAL TO COIL TRANSITION

This example discloses that low concentrations of APC are sufficient to increase HCT and decrease discrimination temperature while reducing the ΔT_d between a perfectly base-paired ODN duplex and an ODN duplex containing a single base-pair mismatch.

The oligonucleotides used in this experiment are those shown in

Example 4. The data displayed in Table 4 show that the discrimination (i.e., ΔT_d) between a perfectly base-paired ODN duplex and an ODN duplex containing a single base-pair mismatch is reduced in a hybridization reaction containing 0.1 M piperidine methanol (PM). Table 4

EXAMPLE 7 APC-BASED BUFFERS REDUCE PCR AMPLIFICATION SPECIFICITY

This example discloses that buffers containing low concentrations of APCs of the present invention are effective in decreasing the hybridization specificity of a polymerase chain reaction (PCR).

The priming efficiency of PCR was examined using the model system described in Rychlik, W., BioTechniques 18(l):84-86, 88-90 1995. DNA of bacteriophage lambda (GenBank Accession No. J02459) was amplified using the 17- mer forward primer (5'-GAACGAAAACCCCCCGC-3') and the 20-mer reverse primer (5'-GATCGCCCCCAAAACACATA-3'), the forward primer being G+C rich. Also, three additional forward primers were synthesized having a G:T mismatch, a G:A mismatch, or a G:G mismatch at the 3' end. Together, the forward and reverse primer pairs amplify a 381-base-pair DNA fragment.

Amplification reactions contained IX amplification buffer (10 mM Tris pH 8.3, 50 mM KC1, 1.5 mM MgCl₂); either 0.1 M 1-ethylpiperidine methanol, pH 7.0 (EPM) or 0.1 M dipropylammonium acetate, pH 6.5; 0.25 ng bacteriophage lambda template DNA; 0.25 ng human genomic DNA (as background DNA to increase overall complexity); 0.8 mM dNTPs; 200 nM of a forward and reverse primer; and 0.5 units Taq DNA polymerase (Perkin Elmer, Norwalk, CT).

The amplification reaction consisted of 25 cycles of the following: 15 seconds at 94°C, 1 minute at 52°C, and 1 minute at 72°C. The amplification products were subjected to electrophoresis through a 2% agarose gel run in 0.5% TBE (45 mM

Tris-borate, pH 8.0; 0.1 mM EDTA). DNA fragments were visualized by staining with ethidium bromide.

The data revealed that PCR products were obtained regardless of the nature of the mismatch. Thus, perfectly base-paired forward primers as well as forward primers having either a G:T mismatch, a G:A mismatch, or a G:G mismatch all yielded the 381 -base-pair bacteriophage lambda DNA fragment when the reactions were performed in the presence of 100 mM EPM.

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference.

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 to be limited by the specific examples provided herein.

Claims

CLAIMS What is claimed is:

1. A composition for reducing the specificity of a hybridization reaction, the composition comprising an oligonucleotide and an annealing promoting compound (APC).

2. The composition of claim 1 wherein the APC is an aminoalcohol the aminoalcohol comprising at least one amine group and at least one hydroxyl group.

3. The composition of claim 2 wherein the aminoalcohol comprises a primary amine.

4. The composition of claim 2 wherein the aminoalcohol comprises a secondary amine.

5. The composition of claim 2 wherein the aminoalcohol comprises a tertiary amine.

6. The composition of claim 2 wherein the aminoalcohol comprises a quaternary amine.

7. The composition of claim 2 wherein the aminoalcohol comprises a primary hydroxyl group.

8. The composition of claim 2 wherein the aminoalcohol comprises a secondary hydroxyl group.

9. The composition of claim 2 wherein the aminoalcohol comprises a tertiary hydroxyl group.

10. The composition of claim 2 wherein the aminoalcohol comprises more than one amine group.

11. The composition of claim 2 or claim 10 wherein the aminoalcohol comprises more than one hydroxyl group.

12. The composition of claim 2, further comprising an acid.

13. The composition of claim 2, further comprising a buffer.

14. The composition of claim 2, further comprising an acid and a buffer.

15. The composition of claim 2, having a pH of between 4 and 10.

16. The composition of claim 15 wherein the pH is between 5 and 9.

17. The composition of claim 15 wherein the pH is between 6 and 8.

18. The composition of claim 2 wherein the aminoalcohol is an aminoalcohol salt.

19. The composition of claim 2 wherein the aminoalcohol is an aminoalcohol salt of an organic acid of the formula R-COOH, where R comprises at least one of H and a carbon-containing organic moiety.

20. The composition of claim 19 wherein R represents a hydrocarbon group.

21. The composition of claim 20 wherein the organic acid is selected from the group consisting of acetic acid, benzoic acid, butyric acid, cyclohexanecarboxyhc acid, decanoic acid, 2-ethylbutyic acid, 2-ethylhexanoic acid, heptanoic acid, hexanoic acid, lauric acid, myristic acid, nonanoic acid, octanoic acid, palmitic acid, and propionic acid.

22. The composition of claim 19 wherein the R group is a halocarbon.

23. The composition of claim 22 wherem the halocarbon comprises a halogen selected from the group consisting of bromide, chloride, fluoride, and iodide.

24. The composition of claim 19 wherein the organic acid is a halogenated carboxylic acid.

25. The composition of claim 24 wherein the halogenated carboxylic acid is selected from the group consisting of chloroacetic acid, dichloroacetic acid, dichlorofluoroacetic acid, difluoroacetic acid, chlorofluoro acetic acid, fluoroacetic acid, trichloroacetic acid, trifluoroacetic acid.

26. The composition of claim 2 wherein the aminoalcohol is an aminoalcohol salt of an inorganic acid.

27. The composition of claim 26 wherein the inorganic acid is selected from the group consisting of hydrochloric acid, phosphoric acid, nitric acid, and hydrobromic acid.

28. The composition of claim 2 wherein the amine group comprises a nitrogen atom that is part of a heterocyclic ring.

29. The composition of claim 28 wherein the heterocyclic ring is selected from the group consisting of aziridine, azetidine, azolidine (pyrrolidine), piperidine (perhydroazine), pyrrole, imidazole, pyridine, pyrimidine, purine, indole, quinoline, isoquinoline, pyrazine, perhydroquinoline, and perhydroisoquinoline.

30. The composition of claim 28 wherein the heterocyclic ring comprises a single nitrogen atom.

31. The composition of claim 28 wherein the heterocyclic ring is piperidine.

32. The composition of claim 2 wherein the aminoalcohol is a piperidine derivative comprising a piperidine ring and at least one hydroxyl group.

33. The composition of claim 32 wherein the hydroxyl group is joined directly to the piperidine ring.

34. The composition of claim 32, comprising a hydrocarbon group disposed between the piperidine ring and the hydroxyl group.

35. The composition of claim 32 wherein the piperidine ring is unsubstituted other than by a hydroxyl group.

36. The composition of claim 32 wherein the piperidine ring is additionally substituted by a hydrocarbon group.

37. The composition of claim 32 wherein the piperidine derivative is selected from the group consisting of 4-hydroxypiperidine, l-methyl-3- piperidinemethanol, 4,4 '-trimethylenebis( 1 -piperidineethanol), 3-piperidinemethanol, l-ethyl-4-hydroxypiperidine, 2-piperidineethanol, 3 -hydroxy- 1-methylpiperidine, 1- ethyl-3-hydoxypiperidine, 4-hydroxy- 1 -methylpiperidine, 1 -methyl-2- piperidinemethanol, 2-piperidinemethanol, and 2,2,6,6-tetramethyl-4-piperidinol.

38. The composition of claim 2 wherein the aminoalcohol is a piperazine derivative comprising a piperazine ring and at least one hydroxyl group.

39. The composition of claim 38, comprising one hydroxyl group wherein the hydroxyl group is joined directly to the piperazine ring.

40. The composition of claim 38, comprising a hydrocarbon group wherein the hydrocarbon group is disposed between the piperazine ring and the hydroxyl group.

41. The composition of claim 38 wherein the piperazine ring comprises a nitrogen atom, the nitrogen atom being unsubstituted.

42. The composition of claim 38 wherein the piperazine ring comprises a nitrogen atom, the nitrogen atom forming a tertiary amine group.

43. The composition of claim 38 wherein the piperazine derivative is selected from the group consisting of l,4-bis(2-hydroxyethyl)piperazine and l-(2- hydroxy ethy l)piperazine .

44. The composition of any one of claims 1-43 wherein the oligonucleotide is immobilized on a solid surface.

45. The composition of claim 44 wherein the solid surface is selected from the group consisting of a nylon tip, a nylon bead, and a nylon membrane.

46. A method of reducing the specificity of a hybridization reaction between two oligonucleotides, the method comprising adding an annealing promoting compound (APC) to the hybridization reaction.

47. A method of reducing the specificity of a hybridization reaction between two oligonucleotides, the method comprising mixing a first oligonucleotide, a second oligonucleotide, and an annealing promoting compound (APC) under conditions suitable for the formation of an oligonucleotide duplex.

48. A method of identifying a target oligonucleotide, the method comprising:

(a) mixing a first oligonucleotide having a sequence complementary to the target oligonucleotide, a second oligonucleotide having a sequence complementary to the complement of the target oligonucleotide, an annealing promoting compound (APC), a polymerase in a buffer compatible with polymerase activity, and a sample containing the target oligonucleotide;

(b) heating the mixture of (a) to a temperature above the melting temperature of the first oligonucleotide and the second oligonucleotide and their respective complementary sequences;

(c) reducing the temperature of the mixture of (b) to below the melting temperature to allow hybridization between the first oligonucleotide, the second oligonucleotide and the target oligonucleotide;

(e) detecting a product of polymerization.

49. The method of claim 48, further comprising:

(f) heating the mixture of (d) to a temperature above the melting temperature of the first oligonucleotide and the second oligonucleotide and their respective complementary sequences; (g) reducing the temperature of the mixture of (f) to below the melting temperature to allow hybridization between the first oligonucleotide, the second oligonucleotide and the target oligonucleotide;

(h) raising the temperature ofthe mixture of (g) to a temperature compatible with polymerase activity.

50. The method of claim 49, further comprising repeating (f), (g) and (h) one or more times.

51. A method comprising performing polymerase chain reaction in the presence of one or more APC.

52. A method of reducing the specificity of a polymerase chain reaction comprising adding an annealing promoting compound (APC) to said polymerase chain reaction.