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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
  • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

• the present invention relates to DNA nucleotide sequence analysis methods and kits used for the same, more particularly, to DNA nucleotide sequence analysis methods and kits for analyzing full length nucleotide sequence of DNA more accurately than the conventional methods through one time analysis of DNA nucleotide sequence analysis.
• Sanger dideoxynucleotide-mediated chain termination method is the conventional method for analyzing DNA nucleotide sequence.
• 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.
• dNTP deoxynucleotide
• ddNTP dideoxynucleotide
• dNTP deoxyguanosinetriphosphate
• dATP deoxyadenosinetri phosphate
• dTTP deoxytymidinetriphosphate
• dCTP deoxycytidinetriphosphate
• ddGTP dideoxyguanosinetriphosphate
• ddATP dideoxyadenosine triphosphate
• ddTTP dideoxytymidinetriphosphate
• ddCTP dideoxycytidinetriphosphate
• 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.
• DNA fragments in various lengths of which the end are terminated with ddNTP are generated.
• 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 .
• 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.
• 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.
• 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.
• the object of the present invention can be achieved by providing a DNA sequence analysis method which employs more than two kinds of DNA polymerases which comprise DNA polymerase of which affinity to ddNTP is higher than that of DNA polymerase which has been used in the conventional Sanger method, and DNA polymerase of which affinity to ddNTP is lower than that of DNA polymerase which has been used in the conventional Sanger method.
• the DNA polymerase of which affinity to ddNTP is higher than that of DNA polymerase used in Sanger method generates relatively short length DNA fragments in large amount
• the DNA polymerase of which affinity to ddNTP is lower than that of DNA polymerase used in Sanger method generates relatively long length DNA fragments in large amount. Therefore, by the method of the present invention which employes concurrently various DNA polymerases of which affinity to ddNTP are different from each other, DNA fragments in various length which come up to 10bps to more than 1,000bps, can be obtained indiscriminately. Consequently, DNA sequence analysis on more longer length of DNA fragments than that can be analyzed by the conventional Sanger method, is possible by the method of the present invention.
• affinity of a DNA polymerase to a dNTP or a ddNTP means that the relative value which represents the extent of contribution of a specific DNA polymerase for the frequency of reaction of ddNTP or dNTP to an end of DNA fragments which are under propagation.
• affinity to ddNTP or dNTP of TopTM DNA polymerase manufactured by BIONEER CORPORATION in Korea is applied as criteria of affinity to ddNTP or dNTP.
• FIG. 1 is the photograph of electrophoresis of DNA fragments generated by using the conventional DNA polymerase of which affinity to dNTP is 3,000 times of affinity to ddNTP.
• FIG. 2 is the photograph of electrophoresis of DNA fragments generated by using the DNA polymerase, of which affinity to dNTP is 0.5 times of affinity to ddNTP.
• FIG. 3 is the photograph of electrophoresis of DNA fragments generated by using the DNA polymerase, of which affinity to dNTP is 8,000 times of affinity to ddNTP.
• FIG. 4 is the photograph of electrophoresis of generated by using the DNA polymerases mixture which comprise concurrently three kinds of DNA polymerases of FIG. 1 to FIG. 3, of which affinities to ddNTP are different from each other.
• FIG. 5 is the photograph of electrophoresis of generated by using the DNA polymerase mixture which comprise concurrently two kinds of various DNA polymerases of FIG. 2 and FIG. 3, of which affinities to ddNTP are different from each other.
• the method of the present invention is characterized in that the DNA polymerase mixture which comprises concurrently the DNA polymerase of which ratio of the affinity to ddNTP to the affinity to dNTP is higher than that of DNA polymerase conventionally used in Sanger method and the DNA polymerase of which ratio of the affinity to ddNTP to the affinity to dNTP is lower than that of DNA polymerase conventionally used in Sanger method, are used.
• the ratio of the affinity to dNTP to the affinity to ddNTP of DNA polymerase may stand for the ratio between the reaction frequency for chain propagation by incorporation of dNTP to DNA fragment terminal catalyzed by the DNA polymerase and the reaction frequency for chain termination by incorporation of ddNTP to DNA fragment terminal catalyzed by the DNA polymerase.
• the method of the present invention comprises : i) a step for the preparation of nucleotide mixture which comprises concurrently the DNA polymerase of which affinity to dNTP is less than 3,000 times, preferably not more than 1,000 times, more preferably not more than 0.5 times of affinity to ddNTP and DNA polymerase of which affinity to dNTP is higher than 3,000 times, preferably not less than 5,000 times, more preferably not less than 8,000 times of affinity to ddNTP; ii) a step for generating complementary DNA fragments by addition of template DNA along with primer into said nucleotide mixture; and iii) a step for recognizing the terminal base of said complementary DNA fragments separated in order of molecular weight to determine the nucleotide sequence of template DNA.
• the DNA sequencing kit is composed of 4 kinds of airtight containers filled with nucleotide mixture along with the DNA polymerase of which affinity to dNTP is less than 3,000 times, preferably not more than 1,000 times, more preferably not more than 0.5 times of the affinity to ddNTP and DNA polymerase of which affinity to dNTP is higher than 3,000 times, preferably not less than 5,000 times, more preferably not less than 8,000 times of the affinity to ddNTP.
• the kit of the present invention comprises : i) an airtight container which contains ddATP, dATP, dGTP, dCTP, dTTP, buffer solution, stabilizer, the DNA polymerase of which affinity to dNTP is less than 3,000 times, preferably not more than 1,000 times, more preferably not more than 0.5 times of the affinity to ddNTP and the DNA polymerase of which affinity to dNTP is higher than 3,000 times, preferably not less than 5,000 times, more preferably not less than 8,000 times of the affinity to ddNTP; ii) an airtight container which contains ddGTP, dATP, dGTP, dCTP, dTTP, buffer solution, stabilizer, the DNA polymerase of which affinity to dNTP is less than 3,000 times, preferably not more than 1,000 times, more preferably not more than 0.5 times of the affinity ddNTP and the DNA polymerase of which affinity to dNTP is higher than 3,000 times
• the mixture of Thermo Sequenase(manufactured by USB company) DNA polymerase of which affinity to dNTP is 0.5 times of the affinity of to ddNTP, and Tfi mutant DNA polymerase (described in Korean Patent Application No.
• nucleotide mixture which contains 3 ⁇ M of dGTP, 30 ⁇ M of dATP, 30 ⁇ M of dTTP, 30 ⁇ M of dCTP and 150nM of ddGTP; the nucleotide mixture which contains 3 ⁇ M of dGTP, 30 ⁇ M of dATP, 30 ⁇ M of dTTP, 30 ⁇ M of dCTP and 1.754 ⁇ M of ddATP; the nucleotide mixture which contains 3 ⁇ M of dGTP, 30 ⁇ M of dATP, 30 ⁇ M of dTTP, 30 ⁇ M of dCTP and 3.02 ⁇ M of ddTTP; and the nucleotide mixture which contains 3 ⁇ M of dGTP, 30 ⁇ M of dATP, 30 ⁇ M of dTTP, 30 ⁇ M of dCTP and l ⁇ M of ddCTP, respectively to prepare the nucleotide mixture which contains 3 ⁇ M of dGTP, 30 ⁇ M of dATP, 30 ⁇ M of d
• TopTM DNA polymerase (produced by Bioneer corporation), of which affinity to dNTP is 3,000 times of the affinity to ddNTP was added into above described four (4) nucleotide mixtures, respectively to prepare the mixtures for generation of complementary DNA fragments for the purpose of comparison with the result of the present invention .
• l-5 ⁇ g of pUC 19 plasmid DNA as template DNA Ml3 Universal Forward 17mer (5 '-gtaaaacgacggccagt, 30pmoles) as primer and distilled water, were added into above- described three(3) kinds of mixtures, respectively to prepare 40 ⁇ g of each reaction mixtures for generating complementary DNA fragments.
• complementary 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 finally proceeded further for 300 seconds at 72°C to make DNA fragment mixtures.
• 40 ⁇ L of Stop solutions (2.5% bromophenolblue, 2.5% xylene cyanol, lOmM NaOH) were added into each DNA fragment mixtures thus obtained to terminate the generation reaction of complementary DNA fragments.
• the DNA fragments thus obtained were separated by electrophoresis in order of the molecular weights 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 of Bioneer corporation) .
• Sequenase and 2.5 units of Ti mutant DNA polymerase and the nucleotide mixture composed of 3 ⁇ M of dGTP, 30 ⁇ M of dATP, 30 ⁇ M of dTTP, 30 ⁇ M of dCTP and 1.754 ⁇ M of ddATP were filled into an airtight container;
• 10X reaction buffer solution 5OOmM Tris-HCl, 20mM MgCl 2 ), 5M of Betain stabilizer, 5 units of the DNA polymerase mixture composed of 2.5 units of Thermo Sequenase 2.5 units of T/i mutant DNA polymerase and the nucleotide mixture composed of 3 ⁇ M of dGTP, 30 ⁇ M of dATP, 30 ⁇ M of dTTP, 30 ⁇ M of dCTP and 3.02 ⁇ M of ddTTP were filled into in an airtight container; and
• FIG. 1 is the photograph of electrophoresis of DNA fragments generated by using the conventional DNA polymerase (TopTM DNA polymerase, 5 units) of which affinity to ddNTP is conventional.
• FIG. 2 is the photograph of electrophoresis of DNA fragments generated by using the DNA polymerase (Tfi mutant DNA polymerase, 5 units )of which affinity to ddNTP is higher than that of conventional DNA polymerase.
• FIG. 3 is the photograph of electrophoresis of DNA fragments generated by using the DNA polymerase (Thermo Sequenase, 5 units), of which affinity to ddNTP is lower than that of conventional DNA polymerase.
• FIG. 4 is the photograph of electrophoresis of generated by using the DNA polymerases mixtures composed of three kinds of DNA polymerases of FIG. 1 to FIG.
• FIG. 5 is the photograph of electrophoresis of generated by using the DNA polymerases mixtures of the present invention composed of two kinds of DNA polymerases of FIG. 2 and FIG. 3(2.5unit, 2.5unit, respectively), which have different affinities to ddNTP.
• 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.

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• 2000
  • 2000-11-25 CN CN 00802714 patent/CN1336960A/zh active Pending
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  • 2000-11-25 WO PCT/KR2000/001354 patent/WO2001038574A1/en not_active Application Discontinuation
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