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    • 73. 发明申请
    • BOOMERAND DNA AMPLIFICATION
    • BOOMERAND DNA扩增
    • WO1993017127A1
    • 1993-09-02
    • PCT/US1993001578
    • 1993-02-19
    • THE STATE OF OREGON acting by and through THE ...
    • THE STATE OF OREGON acting by and through THE ...AHERN, Kevin, G.
    • C12Q01/68
    • C12N15/10C07K14/415C12Q1/6853C12Q2525/307C12Q2525/301C12Q2521/501
    • Methods for amplifying DNA sequences of interest are disclosed. The methods can be performed using only one primer and are also useful in cloning protocols and for sequencing large DNAs. The methods comprise cleaving a sample DNA using an agent, such as a restriction endonuclease, that produces discrete DNA fragments; ligating the fragments to ''adapter'' polynucleotides having a ligatable end and first and second self-complementary sequences separated by a spacer sequence, thereby forming ligated duplexes; denaturing the ligated duplexes to form templates; annealing molecules of an oligonucleotide primer to the templates, the primers being homologous to a primer target site associated with the sequence of interest; extending the primers using a DNA polymerizing agent to form duplex products; and denaturing the duplex products. Subsequent multiple cycles of annealing primers, extending the primers, and denaturing duplex products are usually performed so as to achieve the desired degree of amplification. Sequencing of large DNAs is performed using multiple rounds of DNA amplification, each round employing a primer homologous with a primer target site in the sequence of interest previously amplified. Cloning is facilitated by including a replication origin and selectable marker in the adapters.
    • 公开了扩增感兴趣的DNA序列的方法。 该方法只能使用一个引物进行,也可用于克隆方案和测序大型DNA。 所述方法包括使用产生离散DNA片段的试剂例如限制性内切核酸酶切割样品DNA; 将片段连接到具有可连接末端的“衔接子”多核苷酸和由间隔序列分隔的第一和第二自身互补序列,从而形成连接的双链体; 使连接的双链体变性以形成模板; 将寡核苷酸引物的退火分子与模板反应,引物与与感兴趣的序列相关的引物靶位点同源; 使用DNA聚合剂扩展引物以形成双链体产物; 并使双相产物变性。 通常进行随后的退火引物,延伸引物和变性双链体的多个循环,以达到所需的扩增程度。 使用多轮DNA扩增进行大DNA的测序,每轮使用与预先扩增的感兴趣序列中的引物靶位点同源的引物。 通过在适配器中包含复制起点和可选标记来促进克隆。