EP0832208A1

EP0832208A1 - Materials and methods for the polymerase chain reaction

Info

Publication number: EP0832208A1
Application number: EP96917513A
Authority: EP
Inventors: Paavo Kai Johannes Kinnunen; Pekka Kristian Mustonen; Juha Kalervo Kere
Original assignee: Helsinki University Licensing Ltd
Current assignee: Helsinki University Licensing Ltd
Priority date: 1995-06-07
Filing date: 1996-06-07
Publication date: 1998-04-01
Also published as: AU6006196A; WO1996040900A1

Abstract

Disclosed are improvements for enzyme-catalyzed reactions involving DNA or RNA, including PCR, which are based on conducting such reactions in the presence of lipids.

Description

MATERIALS AND METHODS FOR THE POLYMERASE CHAIN REACTION

FIELD OF THE INVENTION The present invention generally relates to materials and methods for the controlled modification of DNA- or RNA-enzyme interactions by coupling such reactions to lipids.

BACKGROUND OF THE INVENTION

Enzymes which act upon DNA or RNA, such as restriction endonucleases and exonucleases, ligases, polymerases, and others, have become widely available for use in research applications requiring modification, copying, and/or activation (e.g. , transcription) of DNA. For example, the development of thermostable polymerases for use in the polymerase chain reaction (hereinafter referred to as "PCR") has greatly facilitated the use of that technique in the amplification of portions of even the most complex genomes. Of interest to the present invention are numerous methods involving DNA or

RNA modification which are improved upon by the invention. In the present invention, methods for DNA or RNA modification include any method in which an enzyme, together with possible auxiliary proteins, acts upon DNA or RNA. For the purpose of providing background to the present invention, a representative example of a method involving DNA modification is presented below.

One of the most successful methods involving the modification of DNA is the PCR. Most commonly, PCR is used for the selective amplification of a discrete region of DNA from a larger DNA template, such as the genome of an organism, or a portion thereof. The PCR technique utilizes the ability of small segments of DNA (primers) to anneal to portions of a larger single-stranded DNA template which are complementary to the primer sequence. A DNA polymerase is then used to extend the primer in the 5' to 3' direction along the template. The result is a DNA strand extended from the primer which is complementary to the template. Two primers are normally employed, one of which binds to a portion of one of the complementary strands of a double-stranded DNA template, and the other of which binds to a portion of the opposite complementary strand of the same template, such that the 3' ends of the primers point toward each other. The two primers are positioned such that extension of each primer generates a new template for the opposite primer (i.e. , the primer which bound to the opposite strand of the original template).

The reaction begins when a piece of DNA which contains the sequence of interest (the template) is denatured (by heating, usually to 94°C) in the presence of a large molar excess of specific primers as described above. Renaturation (by cooling, usually at 37- 72°C) results in binding of the primers to the template. A thermostable DNA polymerase and a mixture of the four common nucleotides are also present. Polymerization, resulting in primer extension, is then allowed to occur at the optimal temperature for the polymerase (72 °C for the most common polymerase, the taq polymerase). Cycles of denaturation, renaturation, and polymerization are then continuously repeated, eventually resulting in a doubling, in each cycle, of the amount of a product roughly equivalent in length and sequence to the discrete region of DNA sought to be amplified. Obviously, the eventual doubling of the product with each cycle results in the production of large amounts of the discrete sequence to be amplified.

The applications of PCR in molecular biology are legion and will not be addressed here. However, it is of interest to the present invention that standard PCR protocols require that the denaturation, renaturation, and polymerization cycles occur at specific temperatures. Those temperatures are primarily determined by the physical chemical properties of the DNA and the polymerase used in the reaction. For example, 94°C is usually used in the denaturing cycle because that is an optimal temperature at which DNA melting occurs. Similarly, renaturation is usually performed at a temperature which allows maximum specific template binding (most commonly 37°C). In addition, DNA polymerases generally have a temperature optimum. If the reaction is carried out at other than optimum temperature, yields are poor. However, even under optimal conditions, PCR may not produce expected yields due to inefficient extension and/or polymerization and the components, especially the polymerase, are expensive. Standard PCR protocols are generally known to those skilled in the art.

Reactions involving DNA modification, such as those described above, are conventionally carried out in aqueous solution in the presence of organic and inorganic salts, buffers, and the required enzymes. The reaction mixtures must be incubated at optimized temperatures for set periods of time in order for the reaction to run to completion. In the case of PCR, it is additionally required that repeated cycling at specific temperatures, as outlined above, be performed. Also of interest to the present invention are lipids and their physical chemical properties. Lipids exhibit characteristic phase behavior which is a function of, inter alia, temperature and ionic concentrations. Lipid phase transitions are accompanied by dramatic alterations in physical organization. It is now firmly established that several proteins bind to lipids in biological membranes and that such binding is a function of the phase state of the membrane lipids. By changing the physical state of the lipids in the bilayer, one may cooperatively regulate the binding, and therefore, the function of proteins which bind to the lipids. Specifically, several DNA-binding proteins have been shown to bind to lipids, with resulting changes in function depending upon the lipid phase state. However, there are no reports describing the effects of lipids on restriction enzymes, polymerases, ligases, or other DNA (or RNA) processing enzymes.

Phospholipids, for example, which are found in all cellular membranes, comprise two very different physical environments, a hydrophobic interior region and a more- complex hydrophilic exterior region. The hydrophobic tails of most phospholipids exist in two fundamental physical states. At higher temperatures the hydrophobic tails are in a fluid state and generally have rotational freedom; while at lower temperatures, phospholipids are more geometrically constrained. Overall, membrane lipids may exist in varying states of order, or phases, which normally depend upon both temperature and the lipid composition. There are four major forms of organization of lipids in a biological environment (i.e. , in the presence of water). The lamellar liquid crystalline phase (LJ is the fluid state normally depicted in representations of biological membranes. The lamellar gel phase L_β) is formed at low temperatures in lipids in which the lamellar structure is possible. The L_β phase is characterized by tight packing and acyl chains which are more highly ordered as compared to the L_α phase. The _β phase is also characterized by a predominance of the all-trans acyl chain configuration, resulting in greater bilayer thickness than in the L_α phase. In the Hexagonal I phase (Hj), lipids are organized in cylinder-like configurations, with the polar head groups facing outward. Finally, the Hexagonal II phase (Hrj) is characterized by a hexagonal array of cylinders, but with the polar head groups facing inward, surrounding a column of water (i.e. , an inverted micelle). The phase adopted by a particular lipid is a function of, inter alia, temperature and the precise mix of lipids present. For example, some lipids, such as unsaturated phospatidylethanolamines, resist bilayer formation and tend toward the H_π configuration. However, the overall configuration of a biological membrane is determined by the sum of phospholipids present, taking into account other factors, such as temperature, ionic strength, and hydration. The kinetic properties of membranes are due, in large part, to the properties of the phospholipids which comprise them. Membrane lipids create a fluid environment in which conformational changes in both membrane proteins and the lipids themselves allow a diversity of reactions to take place at the membrane which cause changes in cellular processes, including growth. The extent of fluidity in the membrane is a function of temperature, as indicated above, and also of the ratio of phospholipid to cholesterol and the extent of saturation of membrane lipids. One of the more important roles of membrane phospholipids occurs in the regulation of binding between membrane-bound receptors and their ligands. The present inventors have determined that DNA binds to sphingosine-containing membranes. In further studies, the inventors have determined that the presence of either pure lipids or lipid mixtures affects reaction speed, yield, specificity, and enzymatic activity with respect to RNA- and DNA-modifying enzymes, including, but not limited to nucleases, ligases, and polymerases. Thus, the present invention, based upon conducting RNA- and/or DNA- modification reactions in the presence of a lipid matrix provides means for conducting reactions' involving RNA or DNA, such as PCR and restriction digests, in a manner which is faster, less expensive, and which provides higher yields as compared to conventional techniques of the art.

SUMMARY OF THE INVENTION The present invention relates to methods and materials for enhancement of reactions involving RNA or DNA modification by conducting such reactions in the presence of lipids. Methods according to the invention result in increased reaction rates, increased yield, and decreased need for reaction components, such as enzyme.

In a preferred embodiment of the invention, a DNA is amplified by an improvement in a standard PCR method, the improvement comprising conducting the PCR in the presence of lipids. The improved method of the invention results in increased speed of the reaction, improved yields, reduced cost due to the fact that less enzyme must be used, and increased reaction specificity and fidelity in copying.

Preferred lipids for use in the present invention comprise a phosphorylcholine moiety attached to a hydrophobic tail. Highly preferred embodiments of the invention include the use of lipids such as didecylphosphatidylcholine, egg yolk phosphatidylcholine, and 1- palmytoyl-2-acetyl phosphatidylcholine in a concentration range from about 10 μM to about

50 μM.

The present invention is also useful in the improvement of the kinetics, specificity, and yield of enzyme-catalyzed reactions. Thus, in a preferred embodiment of the invention, enzymatic reactions catalyzed by nucleotide kinases and/or phosphatases are improved by conducting such reactions in the presence of a lipid suspension or matrix.

Materials and methods of the invention also enable PCR to be conducted at lower temperatures than are reported in the art and such methods do not require the use of a thermostable polymerase. Upon consideration of the present invention, the skilled artisan understands that the invention may be applied to any method in which RNA or DNA is acted upon by enzymes.

Based upon the principles of the invention, it is understood that conducting such reactions in the presence of a lipid suspension or matrix increases the speed and specificity of such reactions and reduces the requirements for enzyme and other materials, resulting in a considerable cost savings. Accordingly, numerous applications of the invention, in addition to those exemplified herein, are readily apparent to the skilled artisan upon consideration of the present application.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows results of a product extension assay using PCR in the presence of lipids.

Figure 2 shows results of a primer extension assay both with and without lipid. Figure 3 shows results of PCR amplification in the presence of didecyl phosphatidylcholine .

DETAILED DESCRIPTION OF THE INVENTION Standard reaction conditions in various enzyme-catalyzed reactions, and especially such reactions involving DNA, are generally suboptimal, resulting in poor yields and reduced specificity. For example, PCR techniques result in errors in the fidelity of amplification, the magnitude of which depends upon the polymerase used and the ionic environment in which the reaction is conducted. See, e.g. , Kunkel, et al , "The Fidelity of DNA Polymerase and the Polymerases used in the PCR" in Polymerase Chain Reaction I: A Practical Approach (McPherson, et al. , eds, 1991). In addition, conventional DNA manipulation techniques are expensive due to the high cost of enzyme, especially DNA polymerases and restriction endonucleases. The present invention overcomes these and other difficulties in the art and provides a technique for greatly improving the speed, efficiency, and fidelity of methods involving the manipulation of DNA, including reductions in cost due to a reduced necessity for starting materials. In a general sense, the present invention improves conventional techniques by tying reactions involved in those techniques to lipids. For purposes of the present invention, a lipid suspension or matrix simply means that reactions are conducted in the presence of lipid, i.e., wherein lipid is added to the reaction volume. Such improvements may be mediated through lipid-protein or lipid-DNA interactions, possibly reducing activation energies; altered dimensionality of the reactions; and other effects, either alone or in combination. Whatever the mechanism, conducting such reactions in the presence of lipids results in enhanced reaction rates, shorter incubation times, improved yields, and altered reaction temperatures.

Methods according to the invention have been successfully carried out using numerous lipids, including didecyl phosphatidylcholine, egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine, sphingomyelin, sphingosyl phosphatidylcholine, and 1- palmitoyl-2-octyl phosphatidylcholine. Both micelle-forming lipids, such as l-palmitoyl-2- acetyl phosphatidylcholine, and liposome-forming lipids, such as didecyl phosphatidylcholine, may be used according to the invention.

The following Example provides an embodiment of the invention. Numerous additional aspects of the invention are apparent upon consideration of the following Example.

EXAMPLE

ENHANCEMENT OF THE PCR BY LIPID COUPLING

A basic PCR technique is reported in United States Patent No. 4,868,202, incorporated by reference herein. Moreover, a basic approach to PCR is briefly outlined above. Accordingly, the skilled artisan is knowledgeable in applying and using the PCR and the details will not be addressed herein except as where necessary to enable practice of the invention.

A series of experiments was conducted to measure the enhancement of PCR by conducting PCR reactions in the presence of lipids. Comparisons were made by determining the reaction yield upon reaction with or without lipids and by measuring the length of polymerase-synthesized DNA with and without lipids. Typical reactions were conducted at a volume of 20 μl, using approximately 50 μg human genomic DNA as a template. Two primers (10 pmol each), GTTAATAGTAATGTCCTCTCTTTC (SEQ ID NO: 1) and ACCTTTAGTTAGATTGATGAAGCC (SEQ ID NO: 2), which correspond to the 3' and 5' ends, respectively, surrounding a unique approximately 82 bp product corresponding to the unique human sequence-tagged site, SWXD178 as reported in Kere, et al. , Genomics, 14: 241- 248 (1992), incorporated by reference herein, were added to the mixture. The template and product sequences are available through the GenBank database under accession number L14983 (HSSWX178) for human chromosome X STS sWXD178. A Tag polymerase (Boehringer, 0.5 U) and 0.25 μM each of dATP, dGTP, dCTP, and dTTP were used for polymerization in 50 mM KC1, 10 mM Tris-HCl, pH 8.6, 1.5 rnM MgCl₂ and reactions were carried out in a programmable thermocycler (MJR Research). Lipid was added prior to initiation of the reaction in final concentrations of 10, 100, or 250 μM as unilamellar liposomes prepared by extrusion through 0.1 μM polycarbonate membranes with a LiposoFast Membrane Homogenizer (Avestin, Canada), as reported in McDonald, et al., Biochem. Biophys. Acta. , 1061: 297-303 (1991), incorporated by reference herein. Standard reactions without lipid were also run in parallel. The lipids used were selected from lipids having a net positive charge at neutral pH, including stearylamine (STA), sphingosine, dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC), alone and in various combinations. The reactions were performed with and without oil overlay with essentially identical result in each case. Each reaction mixture (i. e. , PCR components with or without lipid) was first heated at 94°C for 150 seconds followed by thirty cycles at 94°C for 30 seconds, 20°C for 45 seconds, and 72°C for 45 seconds. The yield of reaction products was determined by including radiolabled deoxycytosine triphosphate in the reaction mixture and lowering the unlabeled dCTP concentration to 0.025 μM. Reaction products were then separated by agarose gel electrophoresis (a 1.5% gel stained with ethidium bromide). Only one band corresponding to the specific product was observed for each lane. The reaction products were excised from the gel and measured in a scintillation counter.

The results of the assays for product yield are presented in Figure 1. As shown in that Figure, the amount of PCR product was increased threefold in the presence of 250 μM DMPC and fourfold in the presence of a 250 μM mixture of sphingosine and DMPC at a 1 :40 ratio. Increase in the yield of reaction product was also observed when a lipid composition comprising about 90% sphingosine or STA in DMPC was added to the reaction mixture; whereas molar ratios of 15% to 80% inhibited the reaction. The presence of lipid also increased the length of single primer extension products in each case in which lipid was used, indicating that more efficient polymerization is occurring during equivalent PCR runs. The foregoing results clearly show that proper addition of lipids in a PCR results in increased yield of product and more efficient polymerization. Primer extension assays were performed under similar conditions. To measure the average length of extended (polymerized) products, only one primer at a time was included in the reaction in the presence of radiolabelled deoxycytosine triphosphate and unlabelled nucleotides at concentrations of 0.25 μM dATP, dGTP, and dTTP and 0.025 μM dCTP and 250 μM sphingosine: stearylamine in a 1: 1 molar ratio. Identical reactions were run without lipid as controls. Reaction products obtained by primer extension were analyzed by agarose gel electrophoresis, blotted onto nylon membranes, and autoradiographed to visualize the relative molecular weights of the reaction products. As shown in Figure 2, the length of primer extension product is increased in the presence of lipids. Figure 2 also shows that improved yields were obtained when extension was performed in the presence of lipid. In Figure 2, lanes 1-6 represent extension products obtained with lipid and lanes 7-12 represent extension products obtained without lipid. As shown in that Figure, primer extension products obtained from a PCR conducted in the presence of lipids have higher molecular weights and have incorporated more of the label (as indicated by intensity), indicating greater primer extension. In a separate experiment, the polymerase chain reaction was run for 30 cycles as indicated above in the presence of 0.2, 0.4, 0.8, 1.6, 3.1, 6.2, 12.5, 25, 50, 100, or 200 μM concentrations of didecylphosphatidylcholine. As shown in Figure 3, optimal amplification was obtained using between 0.2 μM and 3.1 μM concentrations of the lipid. Three separate controls were run and results showed that PCR product isolated was about 1/10 of that isolated using between about 0.2 μM and about 3.1 μM concentrations of didecylphosphatidylcholine. The results reported above show that the use of lipids in the PCR results in higher yields of PCR products and greater fidelity of amplification. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Helsinki University Licensing Ltd Oy

(ii) TITLE OF INVENTION: "Materials and Methods For The Polymerase Chain Reaction"

(iii) NUMBER OF SEQUENCES: 2

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTTAATAGTA ATGTCCTCTC TTTC 24

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ACCTTTAGTT AGATTGATGA AGCC 24

Claims

CLAIMSWhat is claimed is:

1. In a polymerase chain reaction method, the improvement comprising the step of conducting said polymerase chain reaction in the presence of a lipid.

2. The improvement according to claim 1, wherein said lipid is selected from the group consisting of didecyl phosphatidylcholine, egg phosphatidylcholine, l-palmitoyl-2-acetyl phosphatidylcholine, dimyristoyl phosphatidylcholine, and lysophosphatidylcholine.

3. The improvement according to claim 1, wherein said lipid is present in solution in a concentration from about 10 μM to about 50 μM.