1. Field of the Invention
The present invention belongs in general to a technical field related to modification of DNA molecules, and relates to a preparation method of labeled DNA and use thereof. More specifically, the present invention relates to a novel preparation method of labeled DNA by substituting an deoxyoligonucleotide sequence of a certain part of at least one of two single-stranded DNAs constructing a certain double-stranded DNA molecule with an deoxyoligonucleotide having at least one labeled nucleotide. Moreover, the present invention relates to use of the labeled DNA molecule for direct cloning and direct probing of DNA fragments, as well as to an immobilization method of said labeled DNA molecule onto a solid support.
2. Description of the Related Art
Labeled DNA molecules are widely used, for example, as a means to specifically detect a certain DNA, a nucleotide sequence of a certain region in the DNA and the like, that is they are used as probes in technical fields such as biochemistry, medicine, health care and the like. As typical preparation methods of such a probe, (a) nick translation method, (b) random primer DNA labeling method, (c) DNA terminal labeling method using T4 polynucleotide kinase, and (d) incorporation method of labeled nucleotides into a PCR product using PCR can be mentioned. However, among those methods, whereas a single labeled nucleotide can be incorporated into a high molecular weight DNA of interest at the terminal of the DNA molecule by (c), the incorporating efficiency becomes low. Thus, (c) has a disadvantage in that it is difficult to improve the sensitivity (specific activity) of the probe. Further, because of the feature of the method to use random primers, the required length of DNA fragment to be labeled in (b) is at least 500 bp (mer) and when using a shorter DNA than those, short fragments tend to be dominantly produced (for example, Harrison B. et al., Anal. Biochem. 1986, 158 (2): 307-315). On the other hand, although with either of (b) and (c) labeled nucleotides can be incorporated into a DNA of interest at a high efficiency to achieve a high specific activity, it is difficult to obtain a probe with a certain strand length by those methods (for example, see Feinberg AP, et al., Anal. Biochem. 1983, 132 (1); 6-13 for (b)). Moreover, (a) is, in a simple form to mention, a method having an action where nicks are made within a double-stranded DNA molecule by cutting it with a suitable endonuclease, the nicked DNA strand is digested with a 5xe2x80x2xe2x86x923xe2x80x2 exonuclease, and the digest is replaced with a new DNA strand using a polymerase activity. Whereas this method allows to increase the specific activity, the incorporation rate of nucleotide labeled through a long-term reaction is decreased in general and it is not easy to obtain a probe with a certain strand length through the method.
In addition, with each of the above-mentioned methods of (a), (b) and (c), as all types of the DNA molecules contained in a DNA containing sample is in general to be the DNA subjected to be labeled, it is essential to individualize the DNA molecules of interest when multiple types of DNA molecules present in the sample.
As a certain embodiment to use the above-mentioned DNA probe, DNA tips can be mentioned. DNA tips (or DNA microarraies) have been proposed to be a potent means for analysis of gene expression, mutation, polymorphism and the like, and some of them have been in their practical use. As preparation processes of such DNA tips, a method where the surface of a solid support is firstly treated to bare a positive electric charge and then a DNA is directly immobilized onto the solid support electrostatically, or a synthetic deoxyoligonucleotide is immobilized onto a solid support through covalent binding, or a DNA is directory synthesized on the surface of a solid support (for example, U.S. Pat. Nos. 4,689,405; 5,744,305) has been known. Although each of these methods has its merits and demerits, they are chosen to suite the purpose of use.
For example, to investigate gene expression, cDNA and a part of which, that is a polynucleotide of approximately 200-300 bp, is used for immobilization, and these polynucleotides are generally prepared by PCR amplification using a genome or cDNA library as its template. Moreover, a method where, when the PCR is conducted, biotin or a primer modified with amino group is used to obtain a DNA with the labeled 5xe2x80x2 terminus, and through the terminus of thus prepared DNA, immobilization of the DNA is conducted has been proposed. However, if a number of labeled DNA are to be prepared by PCR to generate a DNA microarray which prerequisite is to align multiple DNA molecules on a solid support, it will be tedious and expensive. Furthermore, because of difference in nature of enzymes (thermostable enzymes) used in PCR method, there is a possibility that some bases in the PCR product (DNA) will be altered, and one must confirm the sequence of said PCR products. In addition, terminal-labeled DNAs prepared with the PCR method have their 5xe2x80x2 terminal labeled. Thus, when a DNA tip is generated using them, it has a structure where its 5xe2x80x2 terminal of the DNA is oriented to the solid surface. In some applications, such a structure is inconvenient. For example, when cDNA synthesis or RNA synthesis, or the following protein synthesis is conducted on a DNA tip, a structure where the 3xe2x80x2 terminus of the DNA is oriented to the solid surface is essential.
On the other hand, when DNAs are directly synthesized on a solid support, conveniently at most only 100 bp of DNA can be immobilized. Moreover, with a method where DNAs are statistically immobilized on a solid support, it is difficult in general to increase the density of the DNA array. In addition, it is difficult to submit thus obtained DNA array for repeating use, because the associating strength of the DNA to the solid support is weak. Moreover, because of the DNA structure which is allowed on the DNA array, stable hybridization is difficult to be achieved.
According to conventional art relating to introduction of a label (or a certain binding group for modification) into a certain region of a polynucleotide, even if it is a prerequisite only to provide a DNA detection probe, only the probe which is likely to reduce the detection accuracy of a target DNA will be obtained due to irregularity in length of the resulting labeled DNA molecules.
Accordingly, it will need to obtain a method where DNA molecules with any length can be selectively labeled or modified, and also where a nucleotide able to specifically label or modify only a certain nucleotide sequence among said molecules with a high efficiency can be provided.
The present inventors have been searching for a method to efficiently modify or label DNAs with a label (which comprises a group or a portion which can form a biological, specific bond, or form a chemical, covalent bond). As a result, it was found that nucleotides which have been labeled with variety types of probes can be efficiently introduced in a certain place of a target DNA in accordance with a reaction which is similar to homologous recombination (or it is called as xe2x80x9cgeneralized recombinationxe2x80x9d) which is considered to progress through complex formation of a single-stranded-DNA with a double-stranded DNA. More specifically, it was found that a target double-stranded DNA fragment (molecule) and a single-stranded DNA (deoxyoligonucleotide) homologous to a nucleotide sequence of a certain region thereof form a triple-stranded DNA, and then when a reaction of a 3xe2x80x2xe2x86x925xe2x80x2 exonuclease is conducted under the existence of four types of dNTPs including at least one labeled dNTP, along with or before a reaction of a 5xe2x80x2xe2x86x923xe2x80x2 polymerase, a certain region of at least one of the two single-stranded DNAs constructing the double-stranded DNA, the region corresponding to the deoxyoligonucleotide, can be replaced by the nucleotide sequence containing the labeled nucleotide. The invention is based on such findings.
Accordingly, the above-mentioned objective can be achieved by a method according to the invention, where a labeled double-stranded DNA molecule is prepared by replacing an deoxyoligonucleotide sequence of a certain region of at least one of the two single-stranded DNAs constructing a certain double-stranded DNA molecule with an deoxyoligonucleotide sequence having at least one labeled (or modified with a binding group), and the labeled single-stranded DNA molecule is separated and obtained if necessary. Such a method comprises the steps of:
(A) incubating at least one type deoxyoligonucleotide having a sequence substantially homologous to said one of the deoxyoligonucleotide sequence of at least one of a certain part with said double-stranded DNA molecule under a condition where said deoxyoligonucleotide and double-stranded DNA molecule can partially form a triple-stranded DNA, and
(B) incubating at least one type of complexes in the presence of four types of dNTPs comprising at least one labeled dNTP under a condition where an deoxyoligonucleotide sequence of a certain region of at least one of the two single-stranded DNAs constituting the double-stranded DNA molecule among the complex can be substituted with the deoxyoligonucleotide sequences having at least one labeled nucleotide (the above method is referred as to a basic preparation method hereinafter).
Moreover, according to the invention, as one of the embodiments where the above-mentioned preparation method is used, when immobilizing DNAs onto a solid support, use of DNAs which are labeled (or modified) with a binding group and can be prepared by the method above is also disclosed. Specifically, a method for immobilization of a target DNA labeled with a member of binding partners onto a solid support on which surface another member of the binding partners is supported by forming a bond between said members, wherein the DNA immobilization method is characterized in that said labeled target DNA is prepared by the steps of:
(A) forming a triple-stranded DNA between a double-stranded DNA consisting of a target DNA and a basic sequence complementary to said DNA, and an deoxyoligonucleotide having a sequence substantially homologous to a part of an deoxyoligonucleotide sequence of the target DNA, and
(B) replacing said deoxyoligonucleotide of a part of the target DNA with an deoxyoligonucleotide having at least one labeled nucleotide in the presence of dNTP comprising at least one dNTP labeled with said member of the binding partners by using thus obtained triple-stranded DNA, is provided (which is referred as to an immobilization method hereinafter).
An deoxyoligonucleotide sequence of a certain region or a part of the deoxyoligonucleotide in said double-stranded DNA may be located at the 3xe2x80x2 terminal side of the double-stranded DNA or in its non-terminal region. As referred in the invention, xe2x80x9cnon-terminal regionxe2x80x9d is, as long as in accordance with objectives of the invention, a region which is not comprise the nucleotide of the both termini of the double-stranded DNA, and in the case of a circular double-stranded DNA it may be any region, and in the case of a straight double-stranded DNA, it means the middle region and other suitable regions which do not includes nucleotides at its both termini.
In the above-mentioned basic preparation method, even under conditions where a certain double-stranded DNA molecule coexists with other double-stranded or single-stranded DNA molecules, substitution of a nucleotide sequence can be conducted selectively using a certain double-stranded DNA molecule as the target. Moreover, according to said preparation method, a nucleotide sequence to be replaced in the double-stranded DNA molecule is substantially restricted to a part corresponding to the deoxyoligonucleotide (a single-stranded DNA) used to form the triple-stranded DNA as a complex. In addition, according to the above-mentioned preparation method, a certain (or target) double-stranded DNA molecule, if its length is longer than that of the deoxyoligonucleotide used to form a triple-stranded DNA, can theoretically replace the above-mentioned nucleotide sequence with any length. Moreover, according to the above-mentioned preparation method, by choosing types or amounts of the labeled dNTP used in the process (B), the labeled double-stranded DNA comprising a desired label at a desired content can be obtained.
Accordingly, as a further embodiment of the invention, a DNA probing composition consisting of a labeled double-stranded DNA molecule obtained in the above-mentioned preparation method or of a labeled single-stranded DNA molecule obtained from said double-stranded DNA molecule; and a composition for direct gene cloning comprising of said labeled double-stranded DNA molecule, and a method to detect DNA fragments are provided.
As a further alternative embodiment of the invention, a detection method of DNA fragments having an deoxyoligonucleotide sequence of a certain region homologous to an deoxyoligonucleotide used in the above-mentioned preparation method which is characterized in that said preparation method is conducted in an aqueous solution comprising a variety of DNA fragments is also provided.
On the other hand, according to the immobilization method, as an other certain embodiment of the invention, a DNA tip where at least one target DNA is immobilized onto a solid support, wherein the DNA tip is formed by a binding between the solid support, on which surface a sort of avidin is supported and the target DNA labeled with a sort of biotin through a complex formation between the biotin and the avidin is also provided.
According to such an immobilization method, by choosing an deoxyoligonucleotide used to form a triple-stranded DNA, only a region of a nucleotide sequence substantially homologous to said deoxyoligonucleotide in a desired region of the target DNA can be specifically replaced with the deoxyoligonucleotide sequence containing at least one nucleotide labeled with a member of the binding partners, and through the member thus introduced into the target DNA the target DNA can be immobilized onto a solid support supporting another member of the binding partners in a desired way.
When such an immobilization method is used, a DNA microarray (or a DNA tip) where a DNA with a desired strand length is immobilized onto a solid support with a desired association strength and an extremely high density can be provided.