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    Mostly natural DNA sequencing by synthesis
    4.
    发明授权
    Mostly natural DNA sequencing by synthesis 有权
    大部分通过合成进行天然DNA测序

    公开(公告)号：US08772473B2

    公开(公告)日：2014-07-08

    申请号：US13262369

    申请日：2010-03-30

    申请人： Xiaohua Huang ,  Eric Roller

    发明人： Xiaohua Huang ,  Eric Roller

    IPC分类号： C07H21/00

    CPC分类号： C12Q1/6869 ,  C12Q2535/119 ,  C12Q2525/301 ,  C12Q2527/137 ,  C12Q2535/113

    摘要： The invention provides a new method for DNA sequencing called “natural sequencing by synthesis” (nSBS). According to the method, DNA that includes a desired sequence is synthesized using a dNTP mix with a small percentage of fluorescently-labeled nucleotides. The fluorescent label is cleavable. In contrast to previous methods that utilize 100% labeled nucleic acids, use of a small percentage of labeled nucleic acids minimizes the distortion of the natural structure of the extending DNA strand and the DNA polymerase. Using the disclosed methods with less than 10,000 copies of template DNA and 10% of the nucleotides labeled, long homopolymer stretches up to 20 bases can be sequenced with high accuracy and Q20 (with 99% accuracy) read lengths of up to 1,000 bases can be achieved. A Q20 read length of greater than 100 bases can potentially be achieved, even if the sequencing is performed with 1,000 copies of a template and 10% of the nucleotides labeled.

    摘要翻译： 本发明提供了一种称为“合成天然测序”（nSBS）的DNA测序的新方法。 根据该方法，使用具有小百分比的荧光标记核苷酸的dNTP混合物合成包含所需序列的DNA。 荧光标记是可切割的。 与使用100％标记的核酸的先前方法相比，使用小百分比的标记的核酸使延伸的DNA链和DNA聚合酶的天然结构的变形最小化。 使用所公开的方法，使用少于10,000拷贝的模板DNA和10％标记的核苷酸，长均聚物延伸多达20个碱基可以高精度测序，Q20（具有99％的准确度）读取长度可达1,000个碱基可以 实现了 即使用1000份拷贝的模板和10％的标记的核苷酸进行测序，潜在地可以实现大于100个碱基的Q20读取长度。

    Identification of alleles
    8.
    发明授权
    Identification of alleles 有权
    鉴定等位基因

    公开(公告)号：US06709816B1

    公开(公告)日：2004-03-23

    申请号：US09419817

    申请日：1999-10-18

    申请人： Xiaohua Huang ,  Thomas B Ryder ,  Paul Kaplan

    发明人： Xiaohua Huang ,  Thomas B Ryder ,  Paul Kaplan

    IPC分类号： C12Q168

    CPC分类号： C12Q1/6827 ,  C12Q2535/125 ,  C12Q2565/501 ,  C12Q2525/161 ,  C12Q2521/131

    摘要： A method for determining the genotype of one or more individuals at a polymorphic locus employs amplification of a region of DNA using primers containing tags and hybridization of the products to one or more probes on a solid support. The genotype or ratio of alleles is identified from the pattern of hybridization. The method can also be used to determine the frequency of different alleles in a population.

    摘要翻译： 用于确定多态性位点处的一个或多个个体的基因型的方法使用含有标签的引物和使用产物与固体支持物上的一种或多种探针杂交来扩增DNA区域。 从杂交模式中鉴定等位基因的基因型或比例。 该方法也可用于确定群体中不同等位基因的频率。

    Single molecule analysis target-mediated ligation of bipartite primers
    9.
    发明授权
    Single molecule analysis target-mediated ligation of bipartite primers 有权
    单分子分析目标介导的两面引物连接

    公开(公告)号：US06316229B1

    公开(公告)日：2001-11-13

    申请号：US09357487

    申请日：1999-07-20

    申请人： Paul M. Lizardi ,  Xiaohua Huang

    发明人： Paul M. Lizardi ,  Xiaohua Huang

    IPC分类号： C12P1934

    CPC分类号： C12Q1/682 ,  C12Q1/6827 ,  C12Q1/6837 ,  C12Q2525/307 ,  C12Q2531/125 ,  C12Q2565/501 ,  C12Q2533/107 ,  C12Q2561/125 ,  C12Q2565/537

    摘要： Disclosed are compositions and a method for detecting single nucleic acid molecules using rolling circle amplification (RCA) of single-stranded circular templates, referred to as amplification target circles, primed by immobilized primers. In one form of the method, referred to as a bipartite primer rolling circle amplification, (BP-RCA), RCA of the amplification target circle (ATC) depends on the formation of a primer by target-mediated ligation. In the presence of a nucleic acid molecule having the target sequence, a probe and a combination probe/primer oligonucleotide can hybridize to adjacent sites on the target sequence allowing the probes to be ligated together. By attaching the first probe to a substrate such as a bead or glass slide, unligated probe/primer can be removed after ligation. The only primers remaining will be primers ligated, via the probe portion of the probe/primer, to the first probe. The ligated primer can then be used to prime replication of its cognate ATC. In this way, an ATC will only be replicated if the target sequence (to which its cognate probe/primer is complementary) is present. BP-RCA is useful, for example, for determining which target sequences are present in a nucleic acid sample, or for determining which samples contain a target sequence. In another form of the method, referred to as immobilized primer rolling circle amplification (IP-RCA), RCA of the ATC depends on incorporation of a target sequence in the ATC during its formation. If the target sequence has been incorporated, a primer that can hybridize to the sequence will prime RCA of the ATC. This form of the method is useful for determining which form or forms of a variable sequence are present in a nucleic acid sample.

    摘要翻译： 公开了使用由固定化引物引发的称为扩增靶圆的单链圆形模板的滚环扩增（RCA）检测单个核酸分子的组合物和方法。 在该方法的一种形式中，称为双极引物滚环扩增（BP-RCA），扩增靶圆环（ATC）的RCA取决于通过靶介导的连接形成引物。 在具有靶序列的核酸分子的存在下，探针和组合探针/引物寡核苷酸可以与目标序列上的相邻位点杂交，使探针连接在一起。 通过将第一探针连接到诸如珠粒或载玻片的基底上，可以在连接后除去未结合的探针/引物。 剩下的唯一引物将是通过探针/引物的探针部分连接到第一探针的引物。 然后连接的引物可用于引发其同源ATC的复制。 以这种方式，如果目标序列（其同源探针/引物互补）存在，ATC将仅被复制。 例如，BP-RCA可用于确定核酸样品中存在哪些靶序列，或用于确定哪些样品含有靶序列。 在该方法的另一种形式中，称为固定化引物滚环扩增（IP-RCA），ATC的RCA取决于ATC在其形成过程中靶序列的结合。 如果靶序列已被并入，则可以与序列杂交的引物将引起ATC的RCA。 该形式的该方法可用于确定核酸样品中存在可变序列的哪种形式或形式。