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    • 9. 发明申请
    • CASCADE NUCLEIC ACID AMPLIFICATION REACTION
    • CASCADE核酸放大反应
    • US20020031764A1
    • 2002-03-14
    • US09465589
    • 1999-12-17
    • JORN ERLAND KOCH
    • C12Q001/68C12P019/34
    • C12Q1/6844C12N15/10C12Q1/6804C12Q1/682C12Q2525/301C12Q2531/125C12Q2543/101C12Q2525/143C12Q2563/131
    • A DNA template consisting of multiple tandem repetitions of an oligonucleotide unit is produced by stepwise copying of an oligonucleotide comprising at least one and a half units of a nucleotide sequence showing dyad symmetry by means of a template- and primer-dependent DNA polymerase in the presence of the necessary nucleoside triphosphates during repeated cycles of denaturation and annealing, or by endless copying of a circular oligonucleotide comprising at least one copy of said oligonucleotide unit by means of a nucleic acid polymerase, which is capable of strand displacement and is substantially without 5null-3null exonuclease activity, in the presence of the necessary nucleoside triphosphates and, if necessary, a primer capable of binding to some portion of the oligonucleotide, or by known reactions. Thereafter, in a cascade nucleic acid amplification reaction, a great number of partial and complete DNA or RNA copies of said DNA template is produced by means of a nucleic acid polymerase, which is capable of strand displacement and is substantially without 5null-3null exonuclease activity, by contacting the template with said nucleic acid polymerase in the presence of the necessary nucleoside triphosphates and, if necessary, a primer capable of binding to the oligonucleotide unit, the polymerase thus synthesizing DNA or RNA originating from, ideally, each repeating oligonucleotide unit in the DNA template. These reactions can be used in methods for detecting a target molecule or group and in processes for the amplification of a particular DNA sequence
    • 由寡核苷酸单位的多重串联重复组成的DNA模板通过在存在下通过模板和引物依赖性DNA聚合酶逐步复制包含至少一个单位的显示二重对称性的核苷酸序列的寡核苷酸来产生 的重复变性和退火循环中必需的核苷三磷酸酯,或通过无限制复制包含至少一个拷贝的所述寡核苷酸单元的环状寡核苷酸,所述核苷酸聚合酶能够进行链置换并且基本上不具有5' -3'核酸外切酶活性,在必需的核苷三磷酸存在下,如果需要,可以与一些部分寡核苷酸结合的引物,或已知的反应。 此后,在级联核酸扩增反应中,通过核酸聚合酶产生大量部分和完整的所述DNA模板的DNA或RNA拷贝,所述核酸聚合酶能够进行链置换并且基本上不含5'-3' 外切核酸酶活性,通过在必需的核苷三磷酸存在下将模板与所述核酸聚合酶接触,并且如果需要,可以与能够结合寡核苷酸单元的引物接触,从而合成来自理想的每一个重复寡核苷酸的DNA或RNA 单元在DNA模板中。 这些反应可用于检测靶分子或基团的方法和用于扩增特定DNA序列的方法