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    Asynchronous primed PCR
    8.
    发明授权
    Asynchronous primed PCR 有权

    公开(公告)号：US06887664B2

    公开(公告)日：2005-05-03

    申请号：US09875211

    申请日：2001-06-05

    申请人： Caifu Chen ,  Michael Egholm ,  Lawrence A. Haff

    发明人： Caifu Chen ,  Michael Egholm ,  Lawrence A. Haff

    IPC分类号： G01N33/53 ,  C12N15/09 ,  C12Q1/68 ,  G01N33/566 ,  C07H21/00 ,  C07H21/02 ,  C12P19/34

    CPC分类号： C12Q1/686 ,  C12Q2561/101 ,  C12Q2527/113 ,  C12Q2527/107

    摘要： An asynchronous thermal cycling protocol for nucleic acid amplification uses two primers with thermal melting temperatures different by about 10 to 30° C. After the higher melting primer has annealed and polymerase mediated extension, the uncopied, single-stranded target sequence may be hybridized and detected by a probe. DNA probes may be cleaved by the exonuclease activity of a polymerase. The probe may be a non-cleaving analog such as PNA. When a probe is labelled with a reporter dye and a quencher selected to undergo energy transfer, e.g. FRET, fluorescence from the reporter dye may be effectively quenched when the probe is unbound. Upon hybridization of the probe to complementary target or upon cleavage while bound to target, the reporter dye is no longer quenched, resulting in a detectable amount of fluorescence. The second, lower-melting primer may be annealed and extended to generate a double-stranded nucleic acid. Amplification may be monitored in real time, including each cycle, or at the end point. The asynchronous PCR thermal cycling protocol can generate a preponderance of the PCR amplicon in single-stranded form by repetition at the end of the protocol of annealing and extension of the higher melting primer.

    Methods for determining sequences information in polynucleotides using mass spectrometry
    9.
    发明授权
    Methods for determining sequences information in polynucleotides using mass spectrometry 失效
    使用质谱法测定多核苷酸序列信息的方法

    公开(公告)号：US5885775A

    公开(公告)日：1999-03-23

    申请号：US726090

    申请日：1996-10-04

    申请人： Lawrence A. Haff ,  Igor Pavlovich Smirnov

    发明人： Lawrence A. Haff ,  Igor Pavlovich Smirnov

    IPC分类号： C12Q1/68 ,  C12P19/34 ,  G01N24/00

    CPC分类号： C12Q1/6886 ,  C12Q1/6858 ,  C12Q2600/16 ,  Y10T436/24

    摘要： The invention relates to methods for determining sequence information in polynucleotides by combining the recent disparate technologies of mass spectrometry and polynucleotide hybridization, amplification, extension and/or ligation techniques. Broadly, in a first step, the method for determining sequence information in a sample polynucleotide includes hybridizing with a sample nucleotide one or a mixture of oligonucleotide probes having a nucleotide sequence complementary to a portion of the sample polynucleotide, thereby forming a complex. Then, in a second step, the complex is contacted with at least a member selected from the group consisting of nucleosides, dideoxynucleosides, polymerases, nucleases, transcriptases, ligases and restriction enzymes to alter at least a subset of said oligonucleotide probes. In a third step, the method provides for determining the molecular weight of at least the subset of altered probes by mass spectrometry and infering the sequence information of the sample polynucleotide therefrom.

    摘要翻译： 本发明涉及通过组合最近不同的质谱技术和多核苷酸杂交，扩增，延伸和/或连接技术来确定多核苷酸序列信息的方法。 广泛地，在第一步中，用于确定样品多核苷酸中的序列信息的方法包括与样品核苷酸1或具有与样品多核苷酸的一部分互补的核苷酸序列的寡核苷酸探针的混合物杂交，从而形成复合物。 然后，在第二步中，将复合物与至少一种选自核苷，双脱氧核苷，聚合酶，核酸酶，转录酶，连接酶和限制酶的成员接触以改变至少一个所述寡核苷酸探针的子集。 在第三步中，所述方法提供通过质谱法确定至少所述改变的探针子集的分子量，并从其中推断样品多核苷酸的序列信息。