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    • 5. 发明授权
    • Method for amplifying and detecting of target nucleic acid sequence
using thermostable enzyme
    • 使用热稳定酶扩增和检测靶核酸序列的方法
    • US5981183A
    • 1999-11-09
    • US821782
    • 1997-03-20
    • Yutaka TakaradaHiroaki InoueShuji ShibataYoshihisa Kawamura
    • Yutaka TakaradaHiroaki InoueShuji ShibataYoshihisa Kawamura
    • C12Q1/68C07H21/04C12P19/34
    • C12Q1/6865
    • In a method for amplifying the specific nucleic acid sequence, a highly specific amplification which has low possibility of non-specific hybridization can be carried out. Highly stable reagents, the activities of which do not decrease in case of supply and storage, are also provided. Thermostable enzymes are used as RNA dependent DNA polymerase, DNA dependent DNA polymerase, DNA dependent RNA polymerase and ribonuclease H, which are required for the amplification system based on replicated RNA. Especially, it is preferable that a thermostable enzyme derived from Thermus thermophilus which has RNA dependent DNA polymerase activity, DNA dependent DNA polymerase activity and ribonuclease H activity, and thermostable DNA dependent RNA polymerase are used together. By this method, inactivation of enzymes are prevented by using thermostable enzymes, and the amplification can be carried out without sequential addition of enzymes.
    • 在扩增特异性核酸序列的方法中,可以进行非特异性杂交的可能性低的高度特异性扩增。 还提供了稳定的试剂,其供应和储存的活性不降低。 热稳定酶用作基于复制RNA的扩增系统所需的RNA依赖性DNA聚合酶,DNA依赖性DNA聚合酶,DNA依赖性RNA聚合酶和核糖核酸酶H。 特别优选共同使用具有RNA依赖性DNA聚合酶活性,DNA依赖性DNA聚合酶活性和核糖核酸酶H活性的嗜热栖热菌的热稳定性酶和耐热性DNA依赖型RNA聚合酶。 通过这种方法,通过使用热稳定酶来防止酶的失活,并且可以不依次加入酶进行扩增。
    • 6. 发明授权
    • Nucleic acid extraction apparatus
    • 核酸提取装置
    • US06281008B1
    • 2001-08-28
    • US09241242
    • 1999-02-01
    • Shigeru KomaiKatsuya DaimonYutaka Takarada
    • Shigeru KomaiKatsuya DaimonYutaka Takarada
    • C12M300
    • B01L3/502B01L3/5082B01L2200/026B01L2300/042B01L2300/046B01L2300/049B01L2400/0487B01L2400/0622B01L2400/0644C12N15/1013G01N1/405G01N35/0098G01N2035/0436Y10S435/82
    • This invention provides an apparatus for extracting nucleic acids from nucleic acid-containing samples. The nucleic acid extraction apparatus of the invention comprises (1) a group of extraction vessels each comprising a reactor tube in which a sample, a reagent solution, and a magnetic carrier are admixed and reacted, a drain cup for pooling an unwanted component solution, and a nucleic acid recovery tube all as secured to a support, (2) a distribution means for introducing a solution into each of the extraction vessels, (3) a stirring means for mixing the solution and magnetic carrier in the reactor tube, (4) a holding means for holding the magnetic carrier stationary within the vessel, (5) a discharging means for discharging the solution from the reactor tube while the magnetic carrier is held stationary, (6) a heating means for heating the solution and magnetic carrier in the reactor tube, and (7) a transfer means for serially transferring the vessels to the given positions.
    • 本发明提供了一种从含核酸样品中提取核酸的装置。 本发明的核酸提取装置包括(1)一组提取容器,每个提取容器均包含反应器管,其中将样品,试剂溶液和磁性载体混合并反应,用于汇集不需要的组分溶液的排水杯, (2)用于将溶液引入每个提取容器的分配装置,(3)用于将溶液和磁性载体混合在反应器管中的搅拌装置,(4) )用于将磁性载体固定在容器内的保持装置,(5)一个放电装置,用于在磁性载体保持静止时将溶液从反应器管排出;(6)加热装置,用于将溶液和磁性载体加热 反应器管,和(7)用于将容器串联转移到给定位置的转移装置。
    • 7. 发明授权
    • Nucleic acid sequence amplification method, detection method, and
reagent kit therefor
    • 核酸序列扩增法,检测方法及试剂盒
    • US5525462A
    • 1996-06-11
    • US875758
    • 1992-04-28
    • Yutaka TakaradaToshiya AonoShuji Shibata
    • Yutaka TakaradaToshiya AonoShuji Shibata
    • C12Q1/68C07H21/04C12P19/34
    • C12Q1/6853
    • An oligonucleotide is provided, which has at least a base sequence A, which is 10-30 nucleotides in length and homologous to a part of a specific nucleic acid sequence to be amplified, and a base sequence B, which is 10-30 nucleotides in length and complementary to a sequence 3' to the part of the specific nucleic acid sequence to be amplified. Sequences A and B are separated by a spacer region of 0-20 nucleotides. The oligonucleotide is used in an amplification method, wherein the oligonucleotide is mixed with a sample containing a specific nucleic acid sequence to be amplified and allowed to anneal to the specific nucleic acid sequence in the sample. The annealed oligonucleotide acts as a primer in an elongation reaction, whereas any nonannealed oligonucleotide is decomposed, except for at least part of the base sequence A. The elongation product is then denatured to a single strand for use as a template to which the remaining oligonucleotide anneals to form a primer for subsequent elongation. Also provided is a detection method, which allows for detection of a specific nucleic acid sequence in a sample, as well as reagent kits for use in the amplification and detection methods.
    • 提供了寡核苷酸,其至少具有碱基序列A,其长度为10-30个核苷酸并且与待扩增的特异性核酸序列的一部分同源;以及碱基序列B,其为10-30个核苷酸 长度并与要扩增的特定核酸序列部分的序列3'互补。 序列A和B被0-20个核苷酸的间隔区隔开。 寡核苷酸用于扩增方法,其中将寡核苷酸与含有待扩增的特异性核酸序列的样品混合并使其与样品中的特定核酸序列退火。 退火的寡核苷酸在伸长反应中用作引物,而除了至少部分碱基序列A之外,任何非退火的寡核苷酸都被分解。然后将延伸产物变性为单链,用作剩余的寡核苷酸 退火以形成随后伸长的底漆。 还提供了一种检测方法,其允许检测样品中的特定核酸序列,以及用于扩增和检测方法的试剂盒。
    • 9. 发明授权
    • Method for amplifying and detecting of target nucleic acid sequence using thermostable enzyme
    • 使用热稳定酶扩增和检测靶核酸序列的方法
    • US06303306B1
    • 2001-10-16
    • US09292435
    • 1999-04-15
    • Yutaka TakaradaHiroaki InoueShuji ShibataYoshihisa Kawamura
    • Yutaka TakaradaHiroaki InoueShuji ShibataYoshihisa Kawamura
    • C12Q168
    • C12Q1/6865C12Q2527/101
    • In a method for amplifying the specific nucleic acid sequence, a highly specific amplification which has low possibility of non-specific hybridization can be carried out. Highly stable reagents, the activities of which do not decrease in case of supply and storage, are also provided. Thermostable enzymes are used as RNA dependent DNA polymerase, DNA dependent DNA polymerase, DNA dependent RNA polymerase and ribonuclease H, which are required for the amplification system based on replicated RNA. Especially, it is preferable that a thermostable enzyme derived from Thermus thermophilus which has RNA dependent DNA polymerase activity, DNA dependent DNA polymerase activity and ribonuclease H activity, and thermostable DNA dependent RNA polymerase are used together. By this method, inactivation of enzymes are prevented by using thermostable enzymes, and the amplification can be carried out without sequential addition of enzymes.
    • 在扩增特异性核酸序列的方法中,可以进行非特异性杂交的可能性低的高度特异性扩增。 还提供了稳定的试剂,其供应和储存的活性不降低。 热稳定酶用作基于复制RNA的扩增系统所需的RNA依赖性DNA聚合酶,DNA依赖性DNA聚合酶,DNA依赖性RNA聚合酶和核糖核酸酶H。 特别优选共同使用具有RNA依赖性DNA聚合酶活性,DNA依赖性DNA聚合酶活性和核糖核酸酶H活性的嗜热栖热菌的热稳定性酶和耐热性DNA依赖型RNA聚合酶。 通过这种方法,通过使用热稳定酶来防止酶的失活,并且可以不依次加入酶进行扩增。
    • 10. 发明授权
    • Reagent for nucleic acid amplification and process for nucleic acid amplification
    • 用于核酸扩增的试剂和用于核酸扩增的方法
    • US06261773B1
    • 2001-07-17
    • US09268710
    • 1999-03-16
    • Masaya SegawaMotohiro KondoYutaka Takarada
    • Masaya SegawaMotohiro KondoYutaka Takarada
    • C12Q168
    • C12Q1/6848C12Q2527/125
    • The present invention provide a process for sequence-specific nucleic acid amplification capable of improving the detection sensitivity and increasing the signal. In particular, the present invention provides a reagent for nucleic acid amplification containing at least one member selected from the group consisting of EDTA, NTA, UDA, CyDTA, DTPA, GEDTA, TTHA and their salts, specifically a reagent for nucleic acid amplification comprising, in addition to the at least one compound, a forward primer having a DNA sequence homologous to a sequence of a target RNA; a reverse primer having a DNA sequence complementary to a sequence of the target RNA and having a promoter for RNA polymerase attached to its 5′ end; ribonucleotides; deoxyribonucleotides; a reverse transcriptase or RNA-directed DNA polymerase; a RNase H; a DNA polymerase or a reverse transcriptase having DNA-directed DNA polymerase activity; and a RNA polymerase. The present invention also provides a process for nucleic acid amplification characterized by carrying out a nucleic acid amplification reaction in the presence of at least one member selected from the group consisting of EDTA, NTA, UDA, CyDTA, DTPA, GEDTA, TTHA and their salts.
    • 本发明提供能够提高检测灵敏度和增加信号的序列特异性核酸扩增方法。 特别地,本发明提供了一种用于核酸扩增的试剂,其包含选自EDTA,NTA,UDA,CyDTA,DTPA,GEDTA,TTHA及其盐中的至少一种,特别是用于核酸扩增的试剂, 除了至少一种化合物之外,具有与靶RNA的序列同源的DNA序列的正向引物; 具有与靶RNA序列互补的DNA序列并具有连接于其5'末端的RNA聚合酶的启动子的反向引物; 核糖核苷酸 脱氧核糖核苷酸; 逆转录酶或RNA指导的DNA聚合酶; 核糖核酸酶H; 具有DNA定向DNA聚合酶活性的DNA聚合酶或逆转录酶; 和RNA聚合酶。 本发明还提供了一种核酸扩增方法,其特征在于在选自EDTA,NTA,UDA,CyDTA,DTPA,GEDTA,TTHA及其盐中的至少一种的存在下进行核酸扩增反应 。