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51. 发明专利

JP2000060554A POLYNUCLEOTIDE PROBE CHIP AND POLYNUCLEOTIDE DETECTION 失效
公开(公告)号：JP2000060554A
公开(公告)日：2000-02-29
申请号：JP24133098
申请日：1998-08-27
申请人： HITACHI LTD
发明人： OKANO KAZUNOBU , KANBARA HIDEKI , UEMATSU SENSHU , MATSUNAGA HIROKO , IRIE TAKASHI , KAJIYAMA TOMOHARU , YASUDA KENJI
IPC分类号： G01N33/53 , B01J19/00 , B01L3/00 , C12N15/09 , C12Q1/68 , C40B40/06 , G01N27/327 , G01N27/447 , G01N37/00
摘要： PROBLEM TO BE SOLVED: To provide a polynucleotide probe chip that holds a many kinds of DNA probes and is useful for detecting a plurality of DNAs. SOLUTION: A polynucleotide probe set comprising a gel precursor having reactive residual groups and a plurality of polynucleotide probes having the residual groups linking to the reactive residue are preliminarily prepared. Arbitrary polynucleotide probes selected from the probe set are combined with individually corresponding gel precursors and the combinations are placed in different small wells 2 on the polynucleotide probe chip 1 and converted to gel. The DNA specimens are forcibly moved by the electrophoresis in the gel. Then, a laser is irradiated from the side face 6 of the chip and the fluorescent light emitted from the whole face of the chips are collectively detected with a high-sensitivity second-dimensional detector. Thus, the DNAs can be detected rapidly in high sensitivity with high hybridization efficiency.

52. 发明专利

JPH11299500A ANALYSIS OF MIXED DNA FRAGMENTS 失效
公开(公告)号：JPH11299500A
公开(公告)日：1999-11-02
申请号：JP11200798
申请日：1998-04-22
申请人： HITACHI LTD
发明人： UEMATSU KAZUMUNE , OKANO KAZUNOBU
IPC分类号： C12Q1/68
摘要： PROBLEM TO BE SOLVED: To analyze a mixture of DNA fragment with increased accuracy by adding a known oligomer to nucleic acid treated with restriction enzymes and subjecting the treated fragments to PCR amplification with the restriction enzyme-recognizing sequence that follows the sequence complementary to the known oligomer and a specific primer. SOLUTION: A specimen of nucleic acid 1 is cleaved with a restriction enzyme into a nucleic acid fragment 2 and an oligomer having a known base sequence 10 is added to the 3'-terminus of the fragment 2. Then, the resultant nucleic acid fragment is used as a template to effect the PCR amplification by using the two kinds of first primer having the first selection base sequence 13 comprising a base sequence complementary to a part or the whole of the recognition sequence in the restriction enzyme following the base sequence of this known oligomer 10 and the 1-3 bases on the 3'-terminus of the base sequence complementary to a part or the whole of the recognition sequence in the restriction enzyme, and the second selection base sequence 11 on the 5'-terminus of the base sequence complementary to the base sequence of the oligomer 10 corresponding to the first selection base sequence and the second primer having the same base sequence as of the second selection base sequence 11 whereby the mixed DNA fragments are analyzed.

53. 发明专利

JPH11299485A METHOD FOR AMPLIFYING NUCLEIC ACID AND AMPLIFICATION PRIMER FOR SAID METHOD 失效
公开(公告)号：JPH11299485A
公开(公告)日：1999-11-02
申请号：JP11200698
申请日：1998-04-22
申请人： HITACHI LTD
发明人： OKANO KAZUNOBU , KANBARA HIDEKI
IPC分类号： G01N33/50 , C12N15/09 , C12Q1/68
摘要： PROBLEM TO BE SOLVED: To surely amplify a nucleic acid by subjecting the part of a specific base sequence or the like to a PCR amplification using a primer having a sequence supplementary to said base sequence excluding at least one base in a specific region from the 3'-terminal and a DNA polymerase. SOLUTION: A group of fragments 35, 39 consisting of base sequence parts having a specific base sequence of a nucleic acid or specific fragments having the specific base sequence of a mixture of fragments of the nucleic acid are prepared by introducing an oligomer 30 having a known base sequence into DNA fragments. Besides a modified primer 31 is prepared by substituting at least one base in a range of the fourth base to the sixth base from the 3'-terminal with a base 36 different from that of the base sequence supplementary to the mold DNA fragment 39 to obtain a primer artificially modified in such a manner that it is not complementary to the mold DNA fragment 35. The group of fragments 35, 39 are subjected to a selective PCR amplification by using said modified primer 31 and a DNA polymerase, and thereby an amplification of a nucleic acid is carried out by suppressing a pseudo positive amplification and a pseudo negative amplification, which are encountered in a case where a group of fragments are classified by using a normal PCR or selective PCR.

54. 发明专利

JPH10174589A DNA ANALYSIS AND REAGENT KIT 失效
公开(公告)号：JPH10174589A
公开(公告)日：1998-06-30
申请号：JP26318697
申请日：1997-09-29
申请人： HITACHI LTD
发明人： MATSUNAGA HIROKO , OKANO KAZUNOBU , KANBARA HIDEKI
IPC分类号： C12N15/09 , C12Q1/68
摘要： PROBLEM TO BE SOLVED: To provide a method for preparing a nested deletion library which does not use cloning and a method for determining a base sequence. SOLUTION: Amplification of each DNA fragment (fragment-1) obtained by cleaving a sample DNA 151 with a restriction enzyme is carried out by using 16 kinds of selection primer sets and DNA 152 (fragment-2) near 5' end of either one of each fragment is obtained by λexonuclease and Klenow fragment. PCR reaction is carried out by using the fragment 2 and the sample DNA 151 to afford a DNA fragment (the third fragment) 158 containing a base sequence complementary to the fragment-2 and longer than the fragment. The starting end of the third fragment is common and the final end is different by a restriction enzyme-cleaving part. The base sequence is determined from the restriction enzyme-cleaving part of each fragment and determination of base sequence in total length of the sample DNA 151 is efficiently carried out.

55. 发明专利

JPH09266800A METHOD FOR PREPARATION OF SPECIMEN AND REAGENT KIT THEREFOR 失效
公开(公告)号：JPH09266800A
公开(公告)日：1997-10-14
申请号：JP27202796
申请日：1996-10-15
申请人： HITACHI LTD
发明人： KANBARA HIDEKI , OKANO KAZUNOBU
IPC分类号： G01N33/53 , C07H21/04 , C12N15/09 , C12Q1/68 , G01N33/566 , G01N33/58
摘要： PROBLEM TO BE SOLVED: To provide a process for the determination of a base sequence of a mixture of DNA fragments by using existing primer. SOLUTION: A DNA oligomer 304 is added to a liquid 320 containing a DNA fragment group 303 obtained by cutting a specimen DNA 1 with NlaIII, a DNA oligomer 304 having a known base sequence is bonded to the 3' terminal of the part cut with the restriction enzyme, the liquid containing the substance bonded to the oligomer is pipetted into 16 tubes, a different primer (having a selecting sequence with a selection function of DNA fragment in the anchor sequence at the 3' and 5' terminals) is put into each tube to effect the complementary strand synthesis reaction and a PCR selection primer necessary for the PCR amplification of the DNA fragment is determined by using a gel electrophoresis spectrum of the synthetic reaction product. The liquid pipetted in the tube 323 is pipetted to vessels of a number equal to the combination number (k) of PCR, a selection primer for PCR is added to each vessel, the PCR amplification is performed by using a primer combination of the anchor primer and the primer set and the obtained DNA fragment is purified and used as a template for the determination of the base sequence. The analysis and the base sequence determination of a DNA can be performed in high efficiency.

56. 发明专利

JPH09149800A ANALYSIS OF DNA AND APPARATUS THEREFOR 失效
公开(公告)号：JPH09149800A
公开(公告)日：1997-06-10
申请号：JP31195095
申请日：1995-11-30
申请人： HITACHI LTD
发明人： KANBARA HIDEKI , OKANO KAZUNOBU
IPC分类号： G01N33/50 , C07H21/04 , C12M1/00 , C12N15/09 , C12Q1/44 , C12Q1/68 , G01N27/447
摘要： PROBLEM TO BE SOLVED: To provide a method for analyzing long DNA by efficient preparation of a sample and an apparatus therefor. SOLUTION: A sample DNA 1 is independently cleaved with two kinds of restriction enzymes and different oligomers are bound to two kinds of DNA fragment groups 7 and 8. The oligomers 10 and 11 are bound to 3' end in the first DNA fragment and bound to 5' end in the second DNA fragment. Two kinds of DNA fragment groups are mixed and DNA polymerase and a complemental strand synthetic substrate are added thereto and complemental strand synthesis is carried out under conditions of cycling reaction using the first primer 30 hybridizing with at least 3' end of first fragment group. The produced DNA chain is used as a template and the complemental strand synthesis is carried out using a primer 31 having substantially same sequence as the oligomer 11 added to 5' end of the second fragment group to prepare a DNA template 52 and sequencing reaction is carried out using the first primer. Thereby, DNA fragment sequence of the first group and a part of DNA fragment sequence of the adjacent to the DNA fragment sequence are measured and linkage of each DNA fragment is obtained using overlapped sequence. Whole base sequence of the sample DNA can efficiently and readily be determined by the method.

57. 发明专利

JPH09300A DETERMINATION OF DNA BASE SEQUENCE AND APPARATUS THEREFOR 失效
公开(公告)号：JPH09300A
公开(公告)日：1997-01-07
申请号：JP14898095
申请日：1995-06-15
申请人： HITACHI LTD
发明人： KANBARA HIDEKI , OKANO KAZUNOBU
IPC分类号： C12N15/09 , C12Q1/68
摘要： PURPOSE: To determine the base sequence of a long DNA successively from the 3'-terminal by using a relatively small number of primers prepared beforehand. CONSTITUTION: A template DNA is converted to a single-stranded DNA, a fraction of the DNA having a prescribed length from the 3' terminal is converted to a double-stranded DNA by a complemental strand synthesis, the double-stranded part is cut with an enzyme, an oligomer is bonded to the cut end to form a new primer-bonded part and the base sequences are successively determined. The determination of the base sequence of a long DNA can be performed with a simple operation in high efficiency in contrast with conventional process necessitating complicate operations and much labor.

58. 发明专利

JPH08173164A PREPARATION OF DNA 失效
公开(公告)号：JPH08173164A
公开(公告)日：1996-07-09
申请号：JP32028894
申请日：1994-12-22
申请人： HITACHI LTD
发明人： KANBARA HIDEKI , OKANO KAZUNOBU , FUJIMORI KIYOSHI
IPC分类号： G01N33/566 , C12N15/09
摘要： PURPOSE: To obtain a DNA through sorting-out PCR amplification without cloning, by fragmenting a double-stranded DNA, linking a known-sequence oligomer to the cut end by ligation, and then by amplifying the resultant DNA fragment with a primer having the sorting-out sequence complementary to the oligomer. CONSTITUTION: A double-stranded DNA is fragmented into plural DNA fragments, an oligomer having a known sequence is linked to one end of each of the fragments and another oligomer with a different known sequence to the other end, and then, complementary chain synthesis of the DNA fragment is conducted by using a primer having an oligonucleotide sequence complementary to the former oligomer and another primer having another oligonucleotide sequence complementary to the latter oligomer and also having sorting-out sequences consisting of a fluorescent label and 1 to 4 bases at 5'and 3'-terminals, respectively, to amplify the number of copies of the DNA fragment, thus easily preparing, without cloning, a DNA specimen to be used for analyzing esp. a long DNA.

59. 发明专利

JPH07116000A FRACTIONATION OF NUCLEIC ACID 失效
公开(公告)号：JPH07116000A
公开(公告)日：1995-05-09
申请号：JP26693693
申请日：1993-10-26
申请人： HITACHI LTD
发明人： KANBARA HIDEKI , OKANO KAZUNOBU , UEMATSU KAZUMUNE
IPC分类号： C12N15/09 , C12Q1/68 , G01N27/447 , G01N33/53 , G01N37/00
摘要： PURPOSE:To simplify the determination of DNA sequences and to save the labor for cloning by fractionating a group of DNA fragments having unknown base sequences based on the differences in their terminal sequences. CONSTITUTION:An oligomers of a known sequence is introduced into the terminals of mixed DNA fragments treated with a restriction enzyme. Supports each fixing an oligomer whihc is complementary to this oligomer and has 2 or 3 arbitrary bases on the 3' chain end are provided. One of the supports is hybridized with the mixed DNA fragments to synthesize the complementary chain to fractionate each fragment. The DNA fragments are used for sequence determination directly or after fractionation.

60. 发明专利

JPH0747000A METHOD FOR CALIBRATING NUCLEIC ACID 失效
公开(公告)号：JPH0747000A
公开(公告)日：1995-02-21
申请号：JP19630993
申请日：1993-08-06
申请人： HITACHI LTD
发明人： NAGAI KEIICHI , KANBARA HIDEKI , OKANO KAZUNOBU , KAWAMOTO KAZUKO , FURUYAMA HIROKO
IPC分类号： C12Q1/68
摘要： PURPOSE:To efficiently calibrate in high precision nucleic acid useful for assaying genetic materials such as virus by immersing e.g. target nucleic acid in a reactional solution containing e.g. nucleic acid base, followed by hybridization, etc., under specified conditions to form a conjugate and detect a labeled substance therein. CONSTITUTION:A target nucleic acid 1 of single-stranded structure and a probe nucleic acid 2 bearing a base sequence serving as fellow key are immersed in a reactional solution containing stock material(s) for supplying dideoxynucleotides having individual nucleic acid bases and individual detectable labeled substances (e.g. fluophor), and both hybridization and extension reaction are induced in the presence of an enzyme having nucleic acid polymerase activity to form a conjugate 4 composed of the probe nucleic acid 2 and the dideoxynucleotide, and a labeled substance taken into the conjugate 4 is detected, thus calibrating the nucleic acid.

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