DNA SEQUENCING METHOD WHICH EMPLOYS VARIOUS NUCLEOTIDE MIXTURES AND KIT USED FOR THE SAME

Abstract: The present invention relates to a DNA nucleotide sequence analysis method which employs dideoxynucleotide-mediated chain termination reaction, and more particularly, directed to a sequencing method for analysis of DNA sequence in more longer length through one step separation process than that can be analyzed by the conventional Sanger method, which is characterized in that DNA fragments are generated by using 2 kinds of nucleotide mixtures of which mol ratios of dideoxynucleotide to deoxynucleotide are different from each other.

Description: DNA SEQUENCING METHOD WHICH EMPLOYS VARIOUS NUCLEOTIDE MIXTURES AND KIT USED FOR THE SAME Technical Field The present invention relates to a DNA nucleotide sequence analysis method which employs dideoxynucleotide- mediated chain termination reaction, and more particularly, directed to a sequencing method for analysis of DNA sequence in more longer length through one step separation process than that can be analyzed by the conventional Sanger method, which is characterized in that DNA fragments are generated by using 2 kinds of nucleotide mixtures of which mol ratios of dideoxynucleotide to deoxynucleotide are different from each other. Background Art As is known, Sanger dideoxynucleotide-mediated chain termination method is the conventional method for analyzing DNA nucleotide sequence. In Sanger method, DNA nucleotide chains propagate through the reaction with deoxynucleotide (dNTP) which contains hydroxyl group substituted at C-3 position of pentose and are terminated through the reaction with dideoxynucleotide (ddNTP) which does not contains hydroxyl group substituted at C-3 position of pentose. In Sanger method, 4 kinds of dNTP, such as deoxyguanosinetriphosphate (dGTP), deoxyadenosinetri phosphate (dATP), deoxytymidinetriphosphate (dTTP) and deoxycytidinetriphosphate (dCTP), are used as substrates which generate DNA fragments complementary to template DNA and 4 kinds of ddNTP, such as dideoxyguanosinetriphosphate (ddGTP), dideoxyadenosinetriphosphate (ddATP), dideoxytymidinetriphosphat (ddTTP) and dideoxycytidinetri phosphate (ddCTP), are used as substrates which terminate chain propagation reaction of complementary DNA fragments. DdNTP does not contain hydroxy group at the C-3 position of pentose, differently from dNTP. Therefore, in case that ddNTP is reacted with the end of complementary DNA fragments which are under propagation, the chain propagation reactions of complementary DNA fragments are terminated. Therefore, in Sanger method, DNA fragments in various lengths of which the end are terminated with ddNTP, are generated. In the Sanger 's method, various kinds of complementary DNA fragments which correspond to the number of nucleotides of template DNA, are generated, and then are separated in order of molecular weight by electrophoresis . Thereafter, the nucleotide sequences of template DNA are recognized by determination of the terminal base of each complementary DNA fragments. However, despite of the convenience of Sanger method, it has been a drawback that DNA in length of only 500 to 700bps can be determined accurately due to the limitation of processivity of complementary DNA propagation reaction. For example, in order to recognize accurately and completely human cDNA of which average length is about 2Kb, DNA sequencing procedure should be repeated more than three times by means of partition of human cDNA. As explained above, Sanger method is time-consuming, very laborious and expensive process to be employed as a sequencing method for DNA in large length. Meanwhile, in the so-called Shot gun method which has been known as a large scale nucleotide sequencing method for genomic DNA, full length DNA is partitioned into several DNA fragments and the sequence of base of each fragments are recognized separately. Thereafter, the sequence of each fragments are compared to each other by using computer, and thereby, full length DNA sequence can be analyzed by deletion of overlapping part. In the above Shot gun method, the time and labor required for analysis of full length DNA sequence can be reduced by means of the expansion of DNA length which can be recognized through one time analysis of DNA sequence. In general, in the conventional Sanger dideoxy nucleotide-mediated chain termination method, single DNA polymerase is employed. Thus, the short DNA fragments which correspond to 20 to 30bps of template DNA and the long DNA fragments which correspond to 600 to 700bps of template DNA, are generated in small amounts, whereas DNA fragments which correspond to 40 to 500bps of template DNA are generated in large amounts. Therefore, the concentration of short DNA fragments and long DNA fragments are relatively low and consequently, the nucleotide sequence of terminal portions of both ends of DNA are difficult to be determined than that of middle portion of DNA. On the reasons of the above, the length of DNA which can be analyzed through one time analysis of nucleotide sequence is limited substantially. Therefore, various researchs for new method which can expand the length of DNA that can be recognized completely through one time analysis of nucleotide sequence by means of more accurate determination of the terminal portions of both ends of template DNA, have been tried for a long time. Disclosure of Invention Therefore, the object of the present invention is to provide a method which can determine more longer sequence of DNA through one time analysis of nucleotide sequence and a kit to be used for the method. The method of the present invention is an improvement of the conventional DNA sequencing method of Sanger, which can be applied for determining more longer DNA than that can be determined by Sanger method. In the conventional DNA sequencing method by means of dideoxynucleotide-mediated chain termination reaction, the nucleotide mixture of which mol ratio of ddNTP to dNTP (hereinafter, the mole ratio of ddNTP to dNTP is represented as "ddNTP/dNTP") is about 0.02, that is ddGTP/dGTP is 0.02 to 0.05; ddATP/dATP is 0.02 to 0.058; ddTTP/dTTP is 0.02 to 0.1; and ddCTP/dCTP is 0.02 to 0.033. The object of the present invention is achieved by providing a DNA sequence analysis method which employs more than two kinds of nucleotide mixtures of which ddNTP/dNTP value are different from each other, i.e., higher than that of the conventional Sanger method or lower than that of the conventional Sanger method. In the present invention, relatively short length DNA fragments are generated by using the nucleotide mixture of which ddNTP/dNTP is higher than that of the conventional Sanger method, that is, ddGTP/dGTP is higher than 0.05, desirably not less than 0.1, more desirably not less than 0.15; ddATP/dATP is higher than 0.058, desirably not less than 0.116, more desirably not less than 0.174; ddTTP/dTTP is higher than 0.1, desirably not less than 0.2, more desirably not less than 0.3; and ddCTP/dCTP is higher than 0.033, desirably not less than 0.066, more desirably not less than 0.099. Also, in the present invention, relatively long length DNA fragments are generated by using the nucleotide mixture of which ddNTP/dNTP is lower than that of the conventional Sanger method, that is, ddGTP/dGTP is less than 0.02, desirably not more than 0.01, more desirably not more than 0.005; ddATP/dATP less than 0.02, desirably not more than 0.116, more desirably not more than 0.0058; ddTTP/dTTP less than 0.02, desirably not more than 0.015, more desirably not more than 0.01; and ddCTP/dCTP is less than 0.02, desirably not more than 0.0066, more desirably not more than 0.0033. Thereafter, DNA fragments in various length thus prepared are separated in order of molecular weight thereof to be determined the terminal base of each DNA fragments . According to the method of the present invention, DNA fragments in various length which come up to 10 bps to more than 1,000bps can be obtained indiscriminately. Consequently, the terminal portion of both end of template DNA can be determined more accurately and completely. Thus, through the method of the present invention, it is possible to analyze the sequence of DNA in more longer length than length of 40 to 500bps that can be analyzed by the conventional Sanger method. In the present invention, the nucleotide mixture of which ddNTP/dNTP is higher than that of the conventional Sanger method, generates relatively short length DNA fragment in a large amount, and the nucleotide mixture of which ddNTP/dNTP is lower than that of the conventional Sanger method, generates relatively long length DNA fragments. Thus DNA fragments in various length correspond to 10 to 1,000bps of template DNA, can be obtained. The DNA fragments in various length thus obtained are mixed together and then, separated through one step process in order of molecular weight thereof to be determined terminal base of each DNA fragments . Therefore, according to the present invention, the sequence of the template DNA of 10 to 1,000bps can be determined more accurately and completely through one step separation process. Brief Description of the Drawings The above objects and other advantages of the present invention will become more apparent by describing in detail a preferred embodiment thereof with reference to the attached drawings, in which: FIG. 1 is the photograph of electrophoresis of DNA fragments generated by using nucleotide mixture of which ddGTP/dGTP is 0.05, ddATP/dATP is 0.058, ddTTP/dTTP is 0.1 and ddCTP/dCTP is 0.033. FIG. 2 is the photograph of electrophoresis of DNA fragments generated by using nucleotide mixture of which ddGTP/dGTP is 0.15, ddATP/dATP is 0.174, ddTTP/dTTP is 0.3 and ddCTP/dCTP is 0.099. FIG. 3 is the photograph of electrophoresis of DNA fragments generated by using nucleotide mixture of which ddGTP/dGTP is 0.005, ddATP/dATP is 0.0058, ddTTP/dTTP is 0.01 and ddCTP/dCTP is 0.0033. FIG. 4 is the photograph of electrophoresis of the mixture of DNA fragments generated according to the methods described in the above explanations of FIG. 1 to FIG. 3. FIG. 5 is the photograph of electrophoresis of the mixture of DNA fragments generated according to the methods described in the above explanations of FIG. 2 to FIG. 3. Best Mode for Carrying Out the Invention Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings . The method of the present invention is characterized in comprising: 1 ) a step for the preparation of the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is higher than 0.05, preferably not less than 0.1, more preferably not less than 0.15; the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is higher than 0.058, preferably not less than 0.116, more preferably not less than 0.174; the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is higher than 0.1, preferably not less than 0.2, more preferably not less than 0.3; and the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is higher than 0.0033, preferably not less than 0.066, more preferably not less than 0.099; 2 ) a step for the preparation of the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is less than 0.02, preferably not more than 0.01, more preferably not more than 0.005; the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is less than 0.02, preferably not more than 0.116, more preferably not more than 0.0058; the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is less than 0.02, preferably not more than 0.015, more preferably not more than 0.01; and the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is less than 0.02, preferably not more than 0.0066, more preferably not more than 0.0033; 3) a step for generating complementary DNA fragments by addition of a template DNA and a primer into said 8 kinds of reaction mixtures prepared in above steps 1 ) and 2 ) , respectively; 4) a step for mixing, separately according to kind of ddNTP, the complementary DNA fragments obtained in step 3) and separating each mixtures of the complementary DNA fragments thus prepared in order of molecular weight; 5 ) a step for determining template DNA nucleotide sequence by recognizing the terminal bases of said complementary DNA fragments thus separated. The kit of the present invention for DNA nucleotide sequencing, comprises 8 kinds of airtight containers composed of: 1) an airtight container of the nucleotide mixture of which mol ratio of ddGTP to dGTP is higher than 0.05, desirably not less than 0.1, more desirably not less than 0.15; 2) an airtight container of the nucleotide mixture of which mol ratio of ddATP to dATP is higher than 0.058, desirably not less than 0.116, more desirably not less than 0.174; 3 ) an airtight container of the nucleotide mixture of which mol ratio of ddTTP to dTTP is higher than 0.1, desirably not less than 0.2, more desirably not less than 0.3; 4 ) an airtight container of the nucleotide mixture of which mol ratio of ddCTP to dCTP is higher than 0.033, desirably not less than 0.066, desirably not less than 0.099; 5 ) an airtight container of the nucleotide mixture of which mol ratio of ddGTP to dGTP is less than 0.02, desirably not more than 0.01, more desirably not more than 0.005; 6) an airtight container of the nucleotide mixture of which mol ratio of ddATP to dATP is less than 0.02, desirably not more than 0.0116, more desirably not more than 0.0058; 7) an airtight container of the nucleotide mixture of which mol ratio of ddTTP to dTTP is less than 0.02, desirably not more than 0.015, more desirably not more than 0.01; and 8) an airtight container of the nucleotide mixture of which mol ratio of ddCTP to dCTP is less than 0.02, desirably not more than 0.0066, more desirably not more than 0.0033. More specifically, the kit of the present invention for DNA nucleotide sequencing comprises 8 kinds of airtight containers composed of: 1) an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dGTP, dATP, dTTP, dCTP and ddGTP of which mol ratio to dGTP is higher than 0.05, desirably not less than 0.1, more desirably not less than 0.15; 2) an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dGTP, dATP, dTTP, dCTP and ddATP of which mol ratio to dATP is higher than 0.058, desirably not less than 0.116, more preferably not less than 0.174; 3) an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dGTP, dATP, dTTP, dCTP and ddTTP of which mol ratio to dTTP is higher than 0.1, desirably not less than 0.2, more desirably not less than 0.3; 4) an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dGTP, dATP, dTTP, dCTP and ddCTP of which mol ratio to dCTP is higher than 0.033, desirably not less than 0.066, desirably not less than 0.099; 5) an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dGTP, dATP, dTTP, dCTP and ddGTP of which mol ratio to dGTP is less than 0.02, desirably not more than 0.01, more desirably not more than 0.005; 6) an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dGTP, dATP, dTTP, dCTP and ddATP of which mol ratio to dATP is less than 0.02, desirably not more than 0.0116, more desirably not more than 0.0058; 7) an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dGTP, dATP, dTTP, dCTP and ddTTP of which mol ratio to dTTP is less than 0.02, desirably not more than 0.015, more desirably not more than 0.01; and 8) an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dGTP, dATP, dTTP, dCTP and ddCTP of which mol ratio to dCTP is less than 0.02, desirably not more than 0.0066, more desirably not more than 0.0033. Hereinafter, the present invention will be described in greater detail with reference to the following examples. The examples are given for illustration of the invention and not intended to be limiting the present invention. Example 1. The Nucleotide mixture of which mol ratio of ddGTP to dGTP is 0.15 by mixing 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 450nM of ddGTP; the nucleotide mixture of which mol ratio of ddATP to dATP is 0.174 by mixing 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 5.262μM of ddATP; the nucleotide mixture of which mol ratio of ddTTP to dTTP is 0.3 by mixing 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 9.06μM of ddTTP; and the nucleotide mixture of which mol ratio of ddCTP to dCTP is 0.099 by mixing 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 3μM of ddCTP, were prepared. In addition, the nucleotide mixture of which mol ratio of ddGTP to dGTP is 0.005 by mixing 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 15nM of ddGTP; the nucleotide mixture of which mol ratio of ddATP to dATP is 0.0058 by mixing 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 175.4nM of ddATP; the nucleotide mixture of which mol ratio of ddTTP to dTTP is 0.01 by mixing 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 0.302μM of ddTTP; and the nucleotide mixture of which mol ratio of ddCTP to dCTP is 0.0033 by mixing 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and O.lμM of ddCTP, were prepared. 10X reaction buffer(500mM Tris-HCl, 20mMMgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, M13 Universal Forward 17mer(5 '-gtaaaacgacggccagt, 30pmoles) as primer, 1.5μg of pUC 19 plasmid DNA as template DNA and distilled water, were added into each nucleotide mixture thus obtained, respectively, to produce 40μg of each reaction mixture for generating DNA fragments. Thereafter, DNA fragments generation reaction were repeated 30 cycles sequentially for 240 seconds at 94 ° C, for 30 seconds at 94°C, for 30 seconds at 50°C, for 60 seconds at 72°C and then proceeded further for 300 seconds at 72°C to make complementary DNA fragments mixture. 40μL of stopping solutions (2.5% bromophenolblue, 2.5% xylene cyanol, lOmM NaOH) were added into the complementary DNA fragments mixtures thus prepared to terminate the generation reaction of complementary DNA fragments. Said DNA fragments thus generated were mixed individually according to kind of dNTP and separated by electrophoresis in order of molecular weight thereof through polyacrylamide gel prepared by 8M Urea and 6% acrylamide. The terminal base of each DNA fragments were recognized by using the silver-staining method (by using silverstar staining kit produced by Bioneer corporation). Example 2. The mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 450nM of ddGTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 5.262nM of ddATP was filled into an airtight container; the mixture of 10X reaction buffer(500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 9.06μM of ddTTP was filled into an airtight container; the mixture of 10X reaction buffer(500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM Of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 3μM of ddCTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 15nM of ddGTP was filled into an airtight container; the mixture of 10X reaction buffer(500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 175.4nM of ddATP was filled into an airtight container; the mixture of 10X reaction buffer(500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 0.302μM of ddTTP was filled into an airtight container; and the mixture of 10X reaction buffer(500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 0. lμM of ddCTP was filled into an airtight container to prepare the DNA sequencing kit of the present invention which is composed of 8 kinds of airtight containers . To each airtight container of the said DNA sequencing kit, 1.5μg of pUC 19 plasmid DNA as template DNA, M13 Universal Forward 17mer (5 '-gtaaaacgacggccagt, 30pmoles) as primer and distilled water were added to produce 40μg of each reaction mixture for generating DNA fragments. The complementary DNA fragments were generated and separated to determine the nucleotide sequence of template DNA, according to the method described in Example 1. Example 3. The mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 450nM of ddGTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 5.262nM of ddATP was filled ino an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 9.06μM of ddTTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 3μM of ddCTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 15nM of ddGTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 175.4nM of ddATP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 0.302μM of ddTTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 0. lμM of ddCTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 150nM of ddGTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 1.754μM of ddATP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM of dATP, 30μM of dTTP, 30μM of dCTP and 3.02μM of ddTTP was filled into an airtight container; the mixture of 10X reaction buffer (500mM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, Top™DNA polymerase, 3μM of dGTP, 30μM Of dATP, 30μM of dTTP, 30μM of dCTP and lμM of ddCTP of which mol ratio of ddCTP to dCTP is 0.033, were filled with an airtight container to produce the DNA nucleotide sequencing kit composed of 12 kinds of airtight containers . To each airtight container of the said DNA sequencing kit, 1.5μg of pUC 19 plasmid DNA as template DNA, M13 Universal Forward 17mer (5 '-gtaaaacgacggccagt, 30pmoles) as primer and distilled water were added to produce 40μg of each reaction mixture for generating DNA fragments . The DNA fragments were generated and separated to determine the nucleotide sequence of template DNA according to the method described in Example 1. FIG. 1 is the photograph of electrophoresis of DNA fragments generated by using nucleotide mixture of which ddGTP/dGTP is 0.05, ddATP/dATP is 0.058, ddTTP/dTTP is 0.1 and ddCTP/dCTP is 0.033, which shows well the complemeatary DNA fragments corresponding to 40 bps to 500bps of template DNA. FIG. 2 is the photograph of electrophoresis of DNA fragments generated by using nucleotide mixture of which ddGTP/dGTP is 0.15, ddATP/dATP is 0.174, ddTTP/dTTP is 0.3 and ddCTP/dCTP is 0.099, which shows well the complemeatary DNA fragments corresponding to 20 bps to 900bps of template DNA. FIG. 3 is the photograph of electrophoresis of DNA fragments generated by using nucleotide mixture of which ddGTP/dGTP is 0.005, ddATP/dATP is 0.0058, ddTTP/dTTP is 0.01 and ddCTP/dCTP is 0.0033, which shows well the complemeatary DNA fragments corresponding to 100 bps to 1000bps of template DNA. FIG. 4 is the photograph of electrophoresis of the mixture of DNA fragments generated according to the methods described in the above explanations of FIG. 1 to FIG. 3, which shows well the complementary DNA fragments corresponding to 20 bps to 1000bps of template DNA. FIG. 5 is the photograph of electrophoresis of the mixture of DNA fragments generated according to the methods described in the above explanations of FIG. 2 to FIG. 3, which shows well the complementary DNA fragments corresponding to 20 bps to 1000bps of template DNA. Industrial Applicability As described in above, The nucleotide sequence of DNA of 10 to 1,000 bps can be analyzed more accurately and completely by method or by using the kit of the present invention than by the conventional Sanger method. Consequently, it is possible to determine DNA sequence in more longer length than that can be determined by Sanger method through one time analysis of nucleotide sequence .

Claims: What is claimed is 1. A DNA nucleotide sequence analsis method which employs the dideoxynucleotide-mediated termination reaction, characterized in comprising : 1 ) a step for the preparation of the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is higher than 0.05; the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is higher than 0.058; the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is higher than 0.1; and the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is higher than 0.0033; 2 ) a step for the preparation of the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is less than 0.02; the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is less than 0.02; the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is less than 0.02; and the reaction mixture for complenentary DNA fragments generation which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is less than 0.02; 3 ) a step for generating complementary DNA fragments by addition of a template DNA and a primer into said 8 kinds of reaction mixtures prepared in above steps 1 ) and 2 ) , respectively; 4) a step for mixing, separately according to kind of ddNTP, the complementary DNA fragments obtained in step 3) and separating each mixtures of the complementary DNA fragments thus prepared in order of molecular weight; 5 ) a step for determining template DNA nucleotide sequence by recognizing the terminal base of said complementary DNA fragments thus separated. 2. The DNA nucleotide sequence analysis method according to Claim 1, wherein the reaction mixtures for complenentary DNA fragments generation, are the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is not less than 0.05; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is not less than 0.116; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is not less than 0.2; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is not less than 0.066; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is not more than 0.01; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is not more than 0.0116; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is not more than 0.015; and the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is not more than 0.0066. 3. The DNA nucleotide sequence analysis method according to Claim 1, wherein the reaction mixtures for complenentary DNA fragments generation, are the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is not less than 0.15; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is not less than 0.174; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is not less than 0.3; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is not less than 0.099; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is not more than 0.005; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is not more than 0.0058; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is not more than 0.01; and the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is not more than 0.0033. 4. The DNA nucleotide sequence analysis method according to Claim 1, wherein the reaction mixtures for complementary DNA fragments generation further comprise the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is 0.02 to 0.05; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is 0.02 to 0.058; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is 0.02 to 0.1; and the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is 0.02 to 0.033. 5. The DNA nucleotide sequence analysis method according to Claim 2, wherein the reaction mixtures for complenentary DNA fragments generation further comprise the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is 0.02 to 0.05; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is 0.02 to 0.058; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is 0.02 to 0.1; and the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is 0.02 to 0.033. 6. The DNA nucleotide sequence analysis method according to Claim 3, wherein the reaction mixtures for complenentary DNA fragments generation further comprise the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddGTP to dGTP is 0.02 to 0.05; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddATP to dATP is 0.02 to 0.058; the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddTTP to dTTP is 0.02 to 0.1; and the reaction mixture which comprises the nucleotide mixture of which mole ratio of ddCTP to dCTP is 0.02 to 0.033. 7. The DNA nucleotide sequence analysis method according to Claim 1, wherein said each mixture of complementary DNA fragments is separated in order of molecular weight through electrophoresis. 8. The DNA nucleotide sequence analysis method according to Claim 1, wherein the the terminal base of complementary DNA fragments are recognized by silver staining method. 9. The DNA nucleotide sequence analysis method according to Claim 2, wherein said each mixture of complementary DNA fragments is separated in order of molecular weight through electrophoresis. 10. The DNA nucleotide sequence analysis method according to Claim 2, wherein the the terminal base of complementary DNA fragments are recognized by silver staining method. 11. The DNA nucleotide sequence analysis method according to Claim 3, wherein said each mixture of complementary DNA fragments is separated in order of molecular weight through electrophoresis. 12. The DNA nucleotide sequence analysis method according to Claim 3, wherein the the terminal base of complementary DNA fragments are recognized by silver staining method. 13. The DNA nucleotide sequence analysis method according to Claim 4, wherein said each mixture of complementary DNA fragments is separated in order of molecular weight through electrophoresis. 14. The DNA nucleotide sequence analysis method according to Claim 4, wherein the the terminal base of complementary DNA fragments are recognized by silver staining method. 15. The DNA nucleotide sequence analysis method according to Claim 5, wherein said each mixture of complementary DNA fragments is separated in order of molecular weight through electrophoresis. 16. The DNA nucleotide sequence analysis method according to Claim 5, wherein the the terminal base of complementary DNA fragments are recognized by silver staining method. 17. The DNA nucleotide sequence analysis method according to Claim 6, wherein said each mixture of complementary DNA fragments is separated in order of molecular weight through electrophoresis. 18. The DNA nucleotide sequence analysis method according to Claim 6, wherein the the terminal base of complementary DNA fragments are recognized by silver staining method. 19. A DNA nucleotide sequencing kit composed of 8 kind of airtight containers composed of: an airtight container filled with the nucleotide mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddGTP of which mole ratio to dGTP is higher than 0.05; an airtight container filled with the nucleotide mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddATP of which mole ratio to dATP is higher than 0.058; an airtight container filled with the nucleotide mixture of reaction buffer, stabilizer, DNA polymerase dATP, dGTP, dCTP, dTTP and ddTTP of which mole ratio to dTTP is higher than 0.1; an airtight container filled with the nucleotide mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddCTP of which mole ratio to dCTP is higher than 0.033; an airtight container filled with the nucleotide mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddGTP of which mole ratio to dGTP is less than 0.02; an airtight container filled with the nucleotide mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddATP of which mole ratio to dATP is less than 0.02; an airtight container filled with the nucleotide mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddTTP of which mole ratio to dTTP is less than 0.02; an airtight container filled with the nucleotide mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddCTP of which mole ratio to dCTP is less than 0.02. 20. The DNA nucleotide sequencing kit according to Claim 19, wherein said 8 kind of airtight containers are filled with, respectively, the nucleotide mixture of which mole ratio of ddGTP to dGTP is not less than 0.1; the nucleotide mixture of which mole ratio of ddATP to dATP is not less than 0.116; the nucleotide mixture of which mole ratio of ddTTP to dTTP is not less than 0.02; the nucleotide mixture of which mole ratio of ddCTP to dCTP is not less than 0.066; the nucleotide mixture of which mole ratio of ddGTP to dGTP is not more than 0.01; the nucleotide mixture of which mole ratio of ddATP to dATP is not more than 0.0116; the nucleotide mixture of which mole ratio of ddTTP to dTTP is not more than 0.015; and the nucleotide mixture of which mole ratio of ddCTP to dCTP is not more than 0.0066. 21. The DNA nucleotide sequencing kit according to Claim 19, wherein said 8 kind of airtight containers are filled with, respectively, the nucleotide mixture of which mole ratio of ddGTP to dGTP is not less than 0.15; the nucleotide mixture of which mole ratio of ddATP to dATP is not less than 0.174; the nucleotide mixture of which mole ratio of ddTTP to dTTP is not less than 0.3; the nucleotide mixture of which mole ratio of ddCTP to dCTP is not less than 0.099; the nucleotide mixture of which mole ratio of ddGTP to dGTP is not more than 0.005; the nucleotide mixture of which mole ratio of ddATP to dATP is not more than 0.0058; the nucleotide mixture of which mole ratio of ddTTP to dTTP is not more than 0.01; and the nucleotide mixture of which mole ratio of ddCTP to dCTP is not more than 0.0033. 22. The DNA nucleotide sequencing kit according to Claim 19, characterized in further comprising: an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddGTP of which mole ratio to dGTP is 0.02 to 0.05; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddATP of which mole ratio to dATP is 0.02 to 0.058; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase dATP, dGTP, dCTP, dTTP and ddTTP of which mole ratio to dTTP is 0.02 to 0.1; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddCTP of which mole ratio to dCTP is 0.02 to 0.033. 23. The DNA nucleotide sequencing kit according to Claim 20, characterized in further comprising: an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddGTP of which mole ratio to dGTP is 0.02 to 0.05; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddATP of which mole ratio to dATP is 0.02 to 0.058; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase dATP, dGTP, dCTP, dTTP and ddTTP of which mole ratio to dTTP is 0.02 to 0.1; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddCTP of which mole ratio to dCTP is 0.02 to 0.033. 24. The DNA nucleotide sequencing kit according to Claim 21, characterized in further comprising: an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddGTP of which mole ratio to dGTP is 0.02 to 0.05; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddATP of which mole ratio to dATP is 0.02 to 0.058; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase dATP, dGTP, dCTP, dTTP and ddTTP of which mole ratio to dTTP is 0.02 to 0.1; an airtight container filled with the mixture of reaction buffer, stabilizer, DNA polymerase, dATP, dGTP, dCTP, dTTP and ddCTP of which mole ratio to dCTP is 0.02 to 0.033.

Inventors: PARK HANOH, YOU JAEHYUNG, PARK, HANOH, YOU, JAEHYUNG
Applicants: BIONEER CORP, BIONEER CORPORATION
Application year: 2000
Main classification: C12Q1
IPC codes: C12M1, C12N15, C12Q1, G01N27