Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-10-12
2003-04-01
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S193000, C536S023100
Reexamination Certificate
active
06541227
ABSTRACT:
BACKGROUND OF THE INVENTION
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 5′→3′ 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 5′ 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 5′ terminal labeled. Thus, when a DNA tip is generated using them, it has a structure where its 5′ 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 3′ 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.
SUMMARY OF THE INVENTION
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 “generalized recombination”) 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 3′→5′ 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 5′→3′ 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
Hattori Atsushi
Shigemori Yasushi
Aisin Seiki Kabushiki Kaisha
Brusca John S.
Burns Doane , Swecker, Mathis LLP
LandOfFree
Preparation of labeled DNA does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Preparation of labeled DNA, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Preparation of labeled DNA will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3010204