Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
    2.
    发明授权
    Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction 有权
    使用连接酶检测反应设计适用于检测核酸序列差异的可寻址阵列的方法

    公开(公告)号:US09340834B2

    公开(公告)日:2016-05-17

    申请号:US13947777

    申请日:2013-07-22

    Abstract: The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range. The present invention further relates to an oligonucleotide array comprising of a support with the plurality of oligonucleotide probes immobilized on the support, a method of using the support to detect single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences, and a kit for such detection, which includes the support on which the oligonucleotides have been immobilized.

    Abstract translation: 本发明涉及一种设计多个捕获寡核苷酸探针的方法,所述多个捕获寡核苷酸探针用于互补寡核苷酸探针与少量错配杂交的载体上,其中多个捕获寡核苷酸探针具有在窄范围内的熔融温度。 本发明还涉及包含固定在载体上的多个寡核苷酸探针的载体的寡核苷酸阵列,使用载体检测多个靶核苷酸序列中的单碱基变化,插入,缺失或易位的方法 和用于这种检测的试剂盒,其包括寡核苷酸已被固定的载体。

    METHOD OF DESIGNING ADDRESSABLE ARRAY SUITABLE FOR DETECTION OF NUCLEIC ACID SEQUENCE DIFFERENCES USING LIGASE DETECTION REACTION

    公开(公告)号:US20130345072A1

    公开(公告)日:2013-12-26

    申请号:US13947777

    申请日:2013-07-22

    Abstract: The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range. The first step of the method involves providing a first set of a plurality of tetramers of four nucleotides linked together, where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides. Groups of 2 to 4 of the tetramers from the first set are linked together to form a collection of multimer units. From the collection of multimer units, all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than 4 times the number of tetramers forming a multimer unit are removed to form a modified collection of multimer units. The modified collection of multimer units is arranged in a list in order of melting temperature. The order of the modified collection of multimer units is randomized in 2° C. increments of melting temperature. Alternating multimer units in the list are then divided into first and second subcollections, each arranged in order of melting temperature. After the order of the second subcollection is inverted, the first collection is linked in order to the inverted second collection to form a collection of double multimer units. From the collection of double multimer units, those units (1) having a melting temperature in ° C. less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption are removed, to form a modified collection of double multimer units. The modified collection of double multimers can be immobilized on a support and used to capture, by hybridization, the products of a ligation detection reaction. As a result, the output of a ligation detection reaction, which is useful in detecting single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences, can be formatted on a support.

    High fidelity thermostable ligase and uses thereof
    8.
    发明授权
    High fidelity thermostable ligase and uses thereof 有权
    高保真热稳定连接酶及其用途

    公开(公告)号:US09127269B2

    公开(公告)日:2015-09-08

    申请号:US14028151

    申请日:2013-09-16

    CPC classification number: C12N9/93

    Abstract: The present invention is directed to a mutant thermostable ligase having substantially higher fidelity than either T4 ligase or Thermus thermophilus ligase. The ligase of the present invention is a mutant of a wild-type thermostable ligase having a histidine adjacent a KXDG motif, where the mutant thermostable ligase has a mutation in its amino sequence where the histidine adjacent the KXDG motif in the wild-type thermostable ligase is replaced with an arginine, and wherein X is any amino acid. The DNA molecule encoding this enzyme as well as expression systems and host cells containing it are also disclosed. The thermostable ligase of the present invention is useful in carrying out a ligase detection reaction process and a ligase chain reaction process.

    Abstract translation: 本发明涉及一种具有比T4连接酶或Thermus thermophilus连接酶具有更高保真度的突变热稳定连接酶。 本发明的连接酶是具有与KXDG基序相邻的组氨酸的野生型热稳定连接酶的突变体,其中突变热稳定连接酶在其氨基序列中具有突变,其中野生型热稳定连接酶中KXDG基序相邻的组氨酸 被精氨酸代替,其中X是任何氨基酸。 还公开了编码该酶的DNA分子以及含有它的表达系统和宿主细胞。 本发明的热稳定连接酶可用于进行连接酶检测反应过程和连接酶链反应过程。

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