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    Purified thermostable enzyme and process for amplifying, detecting, and/or cloning nucleic acid sequences using said enzyme
    6.
    发明公开
    Purified thermostable enzyme and process for amplifying, detecting, and/or cloning nucleic acid sequences using said enzyme 失效
    纯化的热稳定性酶和用于增强，用于检测和/或用于核酸序列的通过使用这种酶的克隆方法。

    公开(公告)号：EP0258017A2

    公开(公告)日：1988-03-02

    申请号：EP87307433.0

    申请日：1987-08-21

    申请人： F. HOFFMANN-LA ROCHE AG

    发明人： Erlich, Henry Anthony ,  Horn, Glenn ,  Saiki, Randall Keichi ,  Stoffel, Susanne ,  Mullis, Kary Banks ,  Lawyer, Frances Cook ,  Gelfand, David Harrow

    IPC分类号： C12N15/10 ,  C12N9/12 ,  C12P19/34 ,  C12N9/96 ,  C12Q1/68

    CPC分类号： C12P19/34 ,  C12N9/1252 ,  C12N9/1276 ,  C12Q1/686 ,  C12Q2527/125

    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 86,000-90,000 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 amplification process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers with a thermostable enzyme to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence, and detecting the sequence so amplified. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed. The enzyme is preferably stored in a buffer of non-ionic detergents that lends stability to the enzyme.

    摘要翻译： 纯化的热稳定酶表中获得确实有独特的特点。 优选地，酶是从栖热菌属aquaticus物种中分离并具有约86.000到90.000道尔顿的分子量。 热塑性表酶可以是天然的或重组，并且可以在温度循环链反应worin至少一个核酸序列在扩增的量从与所选择的引物和三磷酸核苷酸的帮助下一个现有的序列来使用。 扩增过程包括处理该核酸的分离的互补链与摩尔过量的两种寡核苷酸引物，与热塑性表酶延伸引物，形成作为用于合成所需核酸序列，并检测该序列模板，其作用互补的引物延伸产物 因此放大。 该反应的步骤可以作为经常需要的和涉及的温度循环以进行杂交，促进酶的活性和形成的杂交体的变性重复。 所述酶优选地存储在非离子型去污剂做借给稳定性酶的缓冲液。

    Process for amplifying and detecting nucleic acid sequences
    9.
    发明公开
    Process for amplifying and detecting nucleic acid sequences 失效
    Verfahren zur Amplifizierung und zum Nachweis vonNukleinsäuresequenzen。

    公开(公告)号：EP0509612A2

    公开(公告)日：1992-10-21

    申请号：EP92201243.0

    申请日：1986-03-27

    申请人： F. HOFFMANN-LA ROCHE AG

    发明人： Mullis, Kary Banks ,  Arnheim, Norman ,  Saiki, Randall Keichi ,  Erlich, Henry Anthony ,  Horn, Glenn Thomas ,  Scharf, Stephen Joel

    IPC分类号： C12Q1/68 ,  C12P19/34

    CPC分类号： C12N15/10 ,  B01J2219/00495 ,  B01J2219/0059 ,  B01J2219/00722 ,  B01L7/52 ,  C07K14/805 ,  C12Q1/6853 ,  C12Q1/6858 ,  C12Q1/686 ,  C12Q1/6883 ,  C12Q1/6886 ,  C12Q1/689 ,  C40B40/06 ,  C40B60/14 ,  C12Q2525/131 ,  C12Q2521/513

    摘要： The present invention provides a process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample or distinguishing between two different nucleic acid sequences in said sample, wherein each nucleic acid sequence to be detected consists of two separate strands, which process comprises amplifying the specific sequence or sequences (if present) by

    (a) treating the sample with one oligonucleotide primer for each of the two strands of each different specific nucleic acid sequence being detected under hybridizing conditions such that for each strand of each different sequence being detected an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to each strand of each specific sequence such that the extension product synthesized from one primer, when it is separated from its complement, serves as a template for synthesis of an extension product of the other primer;
    (b) treating the product of step (a) under denaturing conditions to separate the primer extension products from their templates;
    (c) treating the product of step (b) with oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template; and
    detecting the thus amplified sequence or sequences (if present).

    摘要翻译： 本发明提供了检测样品中至少一种特定核酸序列的存在或不存在或区分所述样品中两种不同核酸序列的方法，其中待检测的每个核酸序列由两条单独的链组成，其中 方法包括通过以下步骤扩增特异性序列或序列（如果存在）：（a）在杂交条件下检测每个不同特异性核酸序列的两条链中的每条链，用一个寡核苷酸引物处理样品，使得对于每个不同序列的每条链 被检测出与各核酸链互补的每个引物的延伸产物被合成，其中所述引物被选择为与每个特定序列的每条链基本互补，使得从一个引物合成的延伸产物 与其补体分开，用作合成延伸的模板 其他引物的离子产物; （b）在变性条件下处理步骤（a）的产物以将引物延伸产物与其模板分离; （c）用寡核苷酸引物处理步骤（b）的产物，使得使用步骤（b）中制备的每条单链合成引物延伸产物作为模板; 并检测由此扩增的序列或序列（如果存在）。