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51. 发明授权

US5789224A Recombinant expression vectors and purification methods for thermus thermophilus DNA polymerase 失效
标题翻译：热嗜热杆菌DNA聚合酶的重组表达载体和纯化方法
公开(公告)号：US5789224A
公开(公告)日：1998-08-04
申请号：US459383
申请日：1995-06-02
申请人： David H. Gelfand , Frances C. Lawyer , Susanne Stoffel
发明人： David H. Gelfand , Frances C. Lawyer , Susanne Stoffel
IPC分类号： C12N9/12
CPC分类号： C12N9/1252 , C12N9/1276
摘要： Recombinant DNA sequences encoding the DNA polymerase activity of Thermus thermophilus can be used to construct recombinant vectors and transformed host cells for production of the activity. T. thermophilus DNA polymerase is an .about.94 kDa protein especially useful in the DNA amplification procedure known as the polymerase chain reaction.
摘要翻译：可以使用编码嗜热栖热菌的DNA聚合酶活性的重组DNA序列构建重组载体和转化的宿主细胞以产生该活性。嗜热链球菌DNA聚合酶是DIFFERENCE 94 kDa蛋白质，特别适用于称为聚合酶链反应的DNA扩增过程。

52. 发明授权

US5491086A Purified thermostable nucleic acid polymerase and DNA coding sequences from pyrodictium species 失效
标题翻译：纯化的热稳定性核酸聚合酶和来自热解物种的DNA编码序列
公开(公告)号：US5491086A
公开(公告)日：1996-02-13
申请号：US62368
申请日：1993-05-14
申请人： David H. Gelfand , Alice M. Wang
发明人： David H. Gelfand , Alice M. Wang
IPC分类号： C12N1/21 , C12N9/12 , C12N9/96 , C12N15/09 , C12N15/55 , C12Q1/68 , C12R1/01 , C12R1/19 , C12N1/19 , C12N15/54 , C12N15/74
CPC分类号： C12N9/1252
摘要： Recombinant DNA sequences encoding the DNA polymerase activity of Pyrodictium species can be used to construct recombinant vectors and transformed host cells for production of the activity. Pyrodictium enzymes for catalyzing 3'.fwdarw.5' exonuclease activity, i.e., proofreading enzymes, are also provided. The Pyrodictium enzymes are useful in DNA amplification procedures and are not irreversibly inactivated by exposure to 100.degree. C. in a polymerase chain reaction.
摘要翻译：编码Pyrodictium物种的DNA聚合酶活性的重组DNA序列可用于构建重组载体和转化的宿主细胞以产生该活性。还提供了用于催化3' - > 5'核酸外切酶活性的Pyrodictium酶，即校正酶。 Pyrodictium酶可用于DNA扩增程序，并且在聚合酶链反应中通过暴露于100℃不会不可逆地灭活。

53. 发明授权

US5455170A Mutated thermostable nucleic acid polymerase enzyme from Thermus species Z05 失效
标题翻译：来自Thermus物种Z05的突变的热稳定性核酸聚合酶
公开(公告)号：US5455170A
公开(公告)日：1995-10-03
申请号：US113531
申请日：1993-08-27
申请人： Richard D. Abramson , David H. Gelfand , I. Lawrence Greenfield
发明人： Richard D. Abramson , David H. Gelfand , I. Lawrence Greenfield
IPC分类号： C12N9/12 , C12N15/54 , C12N15/70 , C12P19/34 , C12Q1/68 , C12Q1/70
CPC分类号： C12N9/1252 , C12N15/70 , C12N9/1276 , C12P19/34 , C12Q1/6827 , C12Q1/6858 , C12Q1/686 , C12Q1/6876 , C12Q1/6881 , C12Q1/6883 , C12Q1/703
摘要： A purified thermostable enzyme is derived from the eubacterium Thermus species Z05. The enzyme has DNA polymerase, activity reverse transcriptase activity, and optionally 5'.fwdarw.3' exonuclease activity. The enzyme can be native or recombinant, and may be used with primers and nucleoside triphosphates in a temperature-cycling chain reaction where at least one nucleic acid sequence is amplified in quantity from an existing sequence.
摘要翻译：纯化的热稳定酶源自嗜热菌菌种Z05。该酶具有DNA聚合酶，活性逆转录酶活性和任选的5'→3'核酸外切酶活性。酶可以是天然的或重组的，并且可以在温度循环链反应中与引物和核苷三磷酸一起使用，其中至少一个核酸序列以现有序列的量被扩增。

54. 发明授权

US5418149A Reduction of non-specific amplification glycosylase using DUTP and DNA uracil 失效
标题翻译：使用DUTP和DNA尿嘧啶还原非特异性扩增糖基化酶
公开(公告)号：US5418149A
公开(公告)日：1995-05-23
申请号：US960362
申请日：1993-01-05
申请人： David H. Gelfand , Shirley Y. Kwok , John J. Sninsky
发明人： David H. Gelfand , Shirley Y. Kwok , John J. Sninsky
IPC分类号： C12N9/00 , C12N9/12 , C12N15/09 , C12Q1/68 , C12P19/34
CPC分类号： C12N9/1276 , C12Q1/6844
摘要： Improved methods for amplifying nucleic acids can reduce non-specific amplification and minimize the effects of contamination of nucleic acid amplification reaction assays due to amplified product from previous amplifications. The methods involve the introduction of unconventional nucleotide bags into the amplification reaction products and treating the products by enzymatic (e.g., glycosylases) and/or physical-chemical means to render the product incapable of acting as a template for subsequent amplifications.
摘要翻译： PCT No.PCT / US91 / 05210 Sec。 371日期：1993年1月5日 102（e）日期1993年1月5日PCT提交1991年7月23日PCT公布。出版物WO92 / 01814 日期1992年2月6日。用于扩增核酸的改进方法可以减少非特异性扩增，并且由于来自先前扩增的扩增产物而使核酸扩增反应测定的污染的影响最小化。所述方法包括将非常规核苷酸袋引入扩增反应产物并通过酶促（例如糖基酶）和/或物理化学方法处理产物以使产品不能充当随后扩增的模板。

55. 发明授权

US5352600A Purified thermostable enzyme 失效
标题翻译：纯化热稳定酶
公开(公告)号：US5352600A
公开(公告)日：1994-10-04
申请号：US971798
申请日：1992-11-05
申请人： David H. Gelfand , Susanne Stoffel
发明人： David H. Gelfand , Susanne Stoffel
IPC分类号： C12N9/12 , C12N15/70 , C12P19/34 , C12Q1/68 , C12Q1/70 , C12N9/00 , C12N15/00
CPC分类号： C12P19/34 , C12N15/70 , C12N9/1252 , C12N9/1276
摘要： A purified thermostable enzyme is obtained that has unique characteristics. Preferably the enzyme is isolated from the Thermus aquaticus species and has a molecular weight of about 86,000-95,000 daltons. The thermostable enzyme may be native or recombinant and may be used in a temperature-cycling chain reaction wherein at least one nucleic acid sequence is amplified in quantity from an existing sequence with the aid of selected primers and nucleotide triphosphates. The enzyme is preferably stored in a buffer containing non-ionic detergents that lends stability to the enzyme.
摘要翻译：获得了具有独特特征的纯化的热稳定酶。优选地，酶从水生栖热菌属物种分离并且具有约86,000-95,000道尔顿的分子量。热稳定酶可以是天然的或重组的，并且可以用于温度循环链式反应，其中借助于所选择的引物和三磷酸核苷酸，从现有序列中至少一个核酸序列的量被扩增。该酶优选储存在含有赋予酶稳定性的非离子型洗涤剂的缓冲液中。

56. 发明授权

US5116750A Selectable fusion protein having aminoglycoside phosphotransferase activity 失效
标题翻译：具有氨基糖苷磷酸转移酶活性的可选择的融合蛋白
公开(公告)号：US5116750A
公开(公告)日：1992-05-26
申请号：US223430
申请日：1988-07-22
申请人： David H. Gelfand , Frances C. Lawyer , Susanne Stoffel
发明人： David H. Gelfand , Frances C. Lawyer , Susanne Stoffel
IPC分类号： C07K14/55 , C12N9/12 , C12N15/63 , C12N15/70
CPC分类号： C12N15/63 , C07K14/55 , C12N15/70 , C12N9/12 , C07K2319/00 , C07K2319/61 , Y10S930/31
摘要： A novel a selectable fusion protein having aminoglycoside phosphotransferase activity is disclosed. The marker comprises the coding sequences for aminoglycoside phosphotransferase I (APH-I) which has been modified and truncated so as to render its use in recombinant vectors more convenient. The modified, truncated sequence (mtAPH-I) gene is capable, upon expression, of conferring resistance to a number of antibiotics on the host. One of these antibiotics, G418, is toxic to eucaryotic as well as procaryotic hosts. Also disclosed are methods of constructing fusion proteins having N-terminal sequences corresponding to a desired peptide sequence, and C-terminal sequences comprising the amino acids encoded by mtAPH-I. The preferred N-terminal sequences are the first 11 amino acids of .beta.-isopropyl malate dehydrogenase, and the first 7 amino acids of yeast enolase.
摘要翻译：公开了一种具有氨基糖苷磷酸转移酶活性的新型可选择融合蛋白。该标记包括已被修饰和截短的氨基糖苷磷酸转移酶I（APH-1）的编码序列，使其在重组载体中的使用更加方便。修饰的截短序列（mtAPH-I）基因在表达时能够赋予宿主对许多抗生素的抗性。其中一种抗生素G418对真核生物以及原核生物有毒性。还公开了构建具有对应于所需肽序列的N末端序列的融合蛋白的方法，以及包含由mtAPH-I编码的氨基酸的C-末端序列。优选的N-末端序列是苹果酸β-异丙酯脱氢酶的前11个氨基酸，酵母烯醇化酶的前7个氨基酸。

57. 发明授权

US5045463A DNA expression vector and use thereof 失效
标题翻译： DNA表达载体及其用途
公开(公告)号：US5045463A
公开(公告)日：1991-09-03
申请号：US287688
申请日：1988-12-19
申请人： Michael A. Innis , David H. Gelfand , James H. Meade
发明人： Michael A. Innis , David H. Gelfand , James H. Meade
IPC分类号： C12N9/34 , C12N15/10 , C12N15/62 , C12N15/70 , C12N15/81 , C12P7/06
CPC分类号： C12N9/2428 , C12N15/10 , C12N15/625 , C12N15/70 , C12N15/81 , C12P7/06 , C07K2319/02 , C07K2319/036 , Y02E50/17
摘要： A gene having a DNA sequence complementary to that of the glucoamylase polypeptide mRNA from a fungal species, preferably Aspergillus awamori, is prepared. The mRNA is an approximately 2.2 kilobase poly A RNA obtained from fungal cells grown under conditions of glucoamylase induction. Reverse transcription of the mRNA provides a glucoamylase probe used to identify genomic digest fragments containing glucoamylase gene regions, which are sequenced to locate the introns and exons. The genomic fragments are spliced together to form a gene having a DNA sequence with altered or deleted introns which codes for fungal glucoamylase protein and is capable, when correctly combined with a cleaved DNA expression vector, of expressing a non-native protein having glucoamylase enzyme activity upon transformation of a host organism by the vector. The host is preferably bacteria or yeast. The transformed yeast host may be used to produce ethanol.
摘要翻译：制备具有与来自真菌种，优选泡盛曲霉的葡糖淀粉酶多肽mRNA的DNA序列互补的DNA序列的基因。 mRNA是从葡糖淀粉酶诱导条件下生长的真菌细胞获得的约2.2千碱基聚A RNA。 mRNA的逆转录提供了用于鉴定含有葡糖淀粉酶基因区域的基因组消化片段的葡糖淀粉酶探针，其被测序以定位内含子和外显子。将基因组片段拼接在一起以形成具有改变或缺失的内含子的DNA序列的基因，其编码真菌葡糖淀粉酶蛋白，并且当与切割的DNA表达载体正确组合时，能够表达具有葡糖淀粉酶酶活性的非天然蛋白质通过载体转化宿主生物体。宿主优选是细菌或酵母。转化的酵母宿主可用于生产乙醇。

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