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1. 发明申请

US20090270601A1 Differential detection of single nucleotide polymorphisms 审中-公开
标题翻译：差异检测单核苷酸多态性
公开(公告)号：US20090270601A1
公开(公告)日：2009-10-29
申请号：US12386595
申请日：2009-04-21
申请人： Steven Albert Benner , Shuichi Hoshika , Nicole Aurora Leal
发明人： Steven Albert Benner , Shuichi Hoshika , Nicole Aurora Leal
IPC分类号： C12P19/34 , C07H21/04
CPC分类号： C12P19/34 , C12Q1/6809 , C12Q2533/101 , C12Q2525/117
摘要： This application claims processes and compositions that enable discovery of single nucleotide polymorphisms (SNPs) and other sequence variation that follows two essentially identical sequences, one a reference, the other a target, as well as SNPs discovered using these processes and compositions. The inventive process comprises preparation of four sets of primers, “T-extendable”, “A-extendable”, “C-extendable”, and “G-extendable”. These primers, when templated on a reference genome, add (respectively) T, A, C, and G to their 3′-ends. The invention also comprises a step where these primer sets are separately bound to complementary sequences on target DNA and, once bound, prime extension reactions using target DNA as the template. If the target DNA directs incorporation of the same nucleotide as the reference DNA, then the T-, A-, C-, and G-extendable primers are extended (respectively) by T, A, C, and G. The architecture of the process distinguishes products from these extensions from products derived if not T, not A, not C and not G (“3N” or “3”, to indicate the other three nucleotides) are not added. Thus, this process discovers differences between the target and reference DNA in the site queried by the primer extension reaction. The distinction makes the two kinds of products either separable or differentially extendable. This distinction is used to disregard products that added T, A, C, and G and to identify the sequence(s) of primers that added not-T, not-A, not-C, and not-G. Further and optionally, information from these sequences identifies loci of the SNPs in an in silico genome.
摘要翻译：本申请要求能够发现单核苷酸多态性（SNP）和遵循两个基本相同的序列的其他序列变异的方法和组合物，一个是参考，另一个靶标，以及使用这些方法和组合物发现的SNP。本发明方法包括制备四组引物“T可延伸”，“可扩展”，“可扩展”和“可扩展”。当在参照基因组上模板时，这些引物将T，A，C和G（分别）添加到它们的3'末端。本发明还包括一个步骤，其中这些引物组分别与靶DNA上的互补序列结合，并且一旦结合，使用靶DNA作为模板进行初始延伸反应。如果目标DNA引导与参考DNA相同的核苷酸引入，则T，A，C和G分别扩增T，A，C和G可延伸的引物。过程将产品从这些扩展区分出来的产品，如果不是T，而不是A，而不是C而不是G（“3N”或“3”，表示其他三个核苷酸）。因此，该过程发现由引物延伸反应查询的位点中的靶标和参照DNA之间的差异。这种区别使得两种产品可分离或差异扩展。这种区别用于忽视添加T，A，C和G的产物，并鉴定未添加-T，而不是A，非-C和非-G的引物序列。进一步和任选地，来自这些序列的信息识别计算机基因组中SNP的位点。

2. 发明授权

US08212020B2 Reagents for reversibly terminating primer extension 有权
标题翻译：可逆终止引物延伸的试剂
公开(公告)号：US08212020B2
公开(公告)日：2012-07-03
申请号：US12383306
申请日：2009-03-23
申请人： Steven Albert Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
发明人： Steven Albert Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
IPC分类号： C07H17/02 , C07H19/04 , C07H19/10 , C07H19/20 , C07H21/04
CPC分类号： C07H19/10 , C07H19/073 , C07H19/14 , C07H19/173 , C07H23/00
摘要： This invention relates to the field of nucleic acid chemistry, more specifically to the field of compositions of matter that comprise triphosphates of modified 2′-deoxynucleosides and oligonucleotides that are formed when these are appended to the 3′-end of a primer, wherein said modifications comprise NH2 moiety attached to their 3′-hydroxyl group and a fluorescent species in a form of a tag affixed to the nucleobase via a linker that can be cleaved. Such compositions and their associated processes enable and improve the sequencing of oligonucleotides using a strategy of cyclic reversible termination, as outlined in U.S. Pat. No. 6,664,079. Most specifically, the invention concerns compositions of matter that are 5′-triphosphates of ribo- and 2′-deoxyribonucleosides carrying detectable tags and oligonucleotides that might be derived from them. The invention also concerns processes wherein a DNA polymerase, RNA polymerase, or reverse transcriptase synthesizes said oligonucleotides via addition of said triphosphates to a primer.
摘要翻译：本发明涉及核酸化学领域，更具体地涉及包含修饰的2'-脱氧核苷的三磷酸的物质组合物领域和当它们附着于引物的3'-末端时形成的寡核苷酸，其中所述修饰包括连接到它们的3'-羟基上的NH 2部分和通过能够被切割的接头固定在核碱基上的标签形式的荧光物质。这样的组合物及其相关方法使用循环可逆终止的策略实现和改进了寡核苷酸的测序，如美国专利No. 6,664,079。最具体地，本发明涉及携带检测标签的核糖核酸和2'-脱氧核糖核苷的5'-三磷酸的物质组合物，所述可检测标签和寡核苷酸可能来源于它们。本发明还涉及其中DNA聚合酶，RNA聚合酶或逆转录酶通过向引物中加入所述三磷酸合成所述寡核苷酸的方法。

3. 发明授权

US08034923B1 Reagents for reversibly terminating primer extension 有权
标题翻译：可逆终止引物延伸的试剂
公开(公告)号：US08034923B1
公开(公告)日：2011-10-11
申请号：US12383776
申请日：2009-03-27
申请人： Steven Albert Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
发明人： Steven Albert Benner , Daniel Hutter , Nicole Aurora Leal , Fei Chen
IPC分类号： C07H21/02 , C07H21/04 , C07H19/04 , C07H19/048 , C07H19/067 , C07H19/073 , C07H19/167 , C07H19/173
CPC分类号： C07H19/10 , C07H19/20 , C07H21/00
摘要： Processes are disclosed that use 3′-reversibly terminated nucleoside triphosphates to analyze DNA for purposes other than sequencing using cyclic reversible termination. These processes are based on the unexpected ability of terminal transferase to accept these triphosphates as substrates, the unexpected ability of polymerases to add reversibly and irreversibly terminated triphosphates in competition with each other, the development of cleavage conditions to remove the terminating group rapidly, in high yield, and without substantial damage to the terminated oligonucleotide product, and the ability of reversibly terminated primer extension products to capture groups. The presently preferred embodiments of the disclosed processes use a triphosphate having its 3′-OH group blocked as a 3′-ONH2 group, which can be removed in buffered NaNO2 and use variants of Taq DNA polymerase, including one that has a replacement (L616A).
摘要翻译：公开了使用3'-可逆终止的核苷三磷酸以分析DNA作为除了使用循环可逆终止的测序之外的目的的方法。这些过程是基于终端转移酶接受这些三磷酸盐作为底物的意想不到的能力，聚合酶意想不到的能力相互添加可逆地和不可逆地终止的三磷酸酯，发展切割条件以迅速去除终止组，高产生并且对终止的寡核苷酸产物没有实质损害，以及可逆终止的引物延伸产物捕获基团的能力。所公开方法的目前优选的实施方案使用其3'-OH基团被阻断作为3'-ONH2基团的三磷酸酯，其可以在缓冲的NaNO 2中除去，并且使用Taq DNA聚合酶的变体，包括具有替换的变体（L616A ）。

4. 发明授权

US08871469B1 Self-avoiding molecular recognition systems in DNA priming 有权
标题翻译：自我回避分子识别系统在DNA启动中
公开(公告)号：US08871469B1
公开(公告)日：2014-10-28
申请号：US12229159
申请日：2008-08-20
申请人： Steven Albert Benner , Shuichi Hoshika , Fei Chen
发明人： Steven Albert Benner , Shuichi Hoshika , Fei Chen
IPC分类号： C12P19/34 , C07H21/04
CPC分类号： C12Q1/6853 , C07H21/04 , C12Q1/6848 , C12Q1/6876 , C12Q2525/117 , C12Q2600/16 , C12Q2531/113 , C12Q2527/107 , C12Q2525/185 , C12Q2537/143 , C12Q2525/101
摘要： This invention concerns self-avoiding molecular recognition systems (SAMRS), compositions that bind to natural DNA and RNA, but do not bind to compositions at sites that incorporate other SAMRS components, and processes dependent on them. Their utility is shown by discoveries that DNA polymerases accept these compositions as primers and templates, where standard triphosphates are added to primers containing SAMRS components, and added opposite to SAMRS components in the template. A critical mass of data are provided in 16 examples to provide first-generation heuristic rules to permit design of SAMRS sequences can be used as primers and templates that are accepted by DNA polymerases. The presently preferred primers are at least 12 nucleotide units in length, and more preferably between 15 and 30 nucleotides in length. Also preferred are chimeric primers that have standard nucleotides in their 5′-segments, and SAMRS nucleotides in their 3′-segments, and in multiplexed priming.
摘要翻译：本发明涉及自回避分子识别系统（SAMRS），与天然DNA和RNA结合但不结合其他SAMRS组分的位点的组合物以及依赖于它们的方法的组合物。通过发现DNA聚合酶接受这些组合物作为引物和模板，其中将标准三磷酸加入到含有SAMRS组分的引物中，并与模板中的SAMRS组分相反地添加，发现其实用性。在16个例子中提供了大量数据，以提供第一代启发式规则，以允许SAMRS序列的设计可以用作DNA聚合酶所接受的引物和模板。目前优选的引物长度为至少12个核苷酸单位，更优选长度为15至30个核苷酸。还优选的是在其5'-节段中具有标准核苷酸的嵌合引物，以及它们的3'-区段中的SAMRS核苷酸以及多重引发。

5. 发明授权

US08354225B1 Amplification of oligonucleotides containing non-standard nucleobases 有权
标题翻译：含有非标准核碱基的寡核苷酸的扩增
公开(公告)号：US08354225B1
公开(公告)日：2013-01-15
申请号：US11371497
申请日：2006-03-09
申请人： Steven Albert Benner , Albert Michael Sismour
发明人： Steven Albert Benner , Albert Michael Sismour
IPC分类号： C12Q1/68 , C12P19/34 , A01N43/04 , A61K31/70 , C07H21/02 , C07H21/04
CPC分类号： C12P19/34 , C12Q1/686 , C12Q2521/101 , C12Q2525/117
摘要： This invention relates to nucleoside, nucleotide, and oligonucleotide analogs that incorporate non-standard nucleobase analogs, defined to be those that present a pattern of hydrogen bonds to a paired nucleobase analog in a complementary strand that is different from the pattern presented by adenine, guanine, cytosine, and thymine. Most specifically, this invention discloses and claims processes for amplifying nucleic acid analogs containing non-standard nucleobases using polymerase chain reactions, and enzymes that perform this amplification.
摘要翻译：本发明涉及包含非标准核碱基类似物的核苷，核苷酸和寡核苷酸类似物，其被定义为与互补链中的配对核碱基类似物呈现氢键图案的不同于腺嘌呤，鸟嘌呤，胞嘧啶和胸腺嘧啶。最具体地，本发明公开并要求使用聚合酶链反应扩增含有非标准核碱基的核酸类似物的方法，以及进行该扩增的酶。

6. 发明授权

US06037120A Recognition of oligonucleotides containing non-standard base pairs 失效
标题翻译：识别含有非标准碱基对的寡核苷酸
公开(公告)号：US06037120A
公开(公告)日：2000-03-14
申请号：US542142
申请日：1995-10-12
申请人： Steven Albert Benner
发明人： Steven Albert Benner
IPC分类号： C07H21/00 , C12P19/34 , C12Q1/68 , C07H21/04
CPC分类号： C07H21/00 , C12P19/34 , C12Q1/68
摘要： The disclosure describes a new method for intermolecular recognition between two molecules, where complementary oligonucleotide strands bind in aqueous solution, where these strains contain non-standard nucleobases that can pair to fit the Watson-Crick geometry in that they involve a monocyclic six membered ring pairing with a fused bicyclic heterocyclic ring system composed of a five member ring fused with a six membered ring, with the orientation of the heterocycles with respect to each other and with respect to the backbone chain analogous to that found in DNA and RNA, but with a pattern of hydrogen bonds holding the base pair together different from that found in the AT and GC base pairs (a "non-standard base pair").
摘要翻译：本公开描述了两种分子之间的分子间识别的新方法，其中互补的寡核苷酸链在水溶液中结合，其中这些菌株含有可以配对以适应沃森 - 克里克几何的非标准核碱基，因为它们涉及单环六元环配对与由六元环稠合的五元环组成的稠合双环杂环体系，杂环相对于彼此和相对于骨架链的取向类似于在DNA和RNA中发现的杂环，但是具有将碱基对保持的氢键的模式与AT和GC碱基对（“非标准碱基对”）中发现的不同。

7. 发明授权

US5965364A Method for selecting functional deoxyribonucleotide derivatives 失效
标题翻译：选择功能性脱氧核糖核苷衍生物的方法
公开(公告)号：US5965364A
公开(公告)日：1999-10-12
申请号：US775402
申请日：1996-12-31
申请人： Steven Albert Benner
发明人： Steven Albert Benner
IPC分类号： C07H21/00 , C12N15/10 , C12P19/34 , C12Q1/68 , G01N33/48
CPC分类号： C07H21/00 , C12N15/102 , C12P19/34 , C12Q1/68 , Y10T436/143333
摘要： Improvements are provided for methods for selecting in vitro compositions of matter that serve as ligands and catalysts. Also provided are novel nucleoside analogs that expand the structural diversity of oligonucleotides and their analogs.
摘要翻译：提供用于选择用作配体和催化剂的物质的体外组合物的方法的改进。还提供了扩增寡核苷酸及其类似物的结构多样性的新型核苷类似物。

8. 发明授权

US09297041B1 Inexpensive autonomous assembly of ultra-large (UL) DNA constructs 有权
标题翻译：超大型（UL）DNA构建体的廉价自主装配
公开(公告)号：US09297041B1
公开(公告)日：2016-03-29
申请号：US14048671
申请日：2013-10-08
申请人： Steven Albert Benner
发明人： Steven Albert Benner
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6862 , C12P19/34 , C12Q2521/501 , C12Q2525/117
摘要： This invention provides processes to assemble many (greater than 20) partially overlapping single stranded DNA molecules (fragments) having preselected sequences, followed by extension of those strands that hybridize at terminal overlap regions, and ligation of the extend products, creating a double-stranded DNA assembly. These processes use non-standard nucleotides carrying heterocyclic nucleobase analogs that implement non-standard hydrogen bonding patterns; these allow controlled annealing of the single stranded fragments via Watson-Crick rules, with less or no interference from a range of non-Watson Crick interactions, hairpin formations, or off-target hybridization displayed by standard nucleobases. This process includes an optional conversion step that replaces non-standard nucleobase pairs with standard nucleobase pairs, generating large synthetic DNA (LS-DNA) molecules containing only natural nucleotides. As useful application, this invention allows the assembly of genes encoding whole proteins (typically 1000-3000 nucleotide pairs) from a collection of single stranded DNA fragments at reduced cost and effort.
摘要翻译：本发明提供了组装具有预选序列的许多（大于20个）部分重叠的单链DNA分子（片段）的方法，随后延伸在末端重叠区域杂交的那些链，以及延伸产物的连接，产生双链 DNA组装。这些方法使用携带杂环核苷酸类似物的非标准核苷酸实现非标准氢键合模式; 这些允许通过Watson-Crick规则对单链片段进行受控退火，具有来自非标准核碱基显示的非沃森克里克相互作用，发夹结构或脱靶杂交范围的较少或不受干扰。该过程包括可选的转化步骤，其用标准核碱基对取代非标准碱基对，产生仅含有天然核苷酸的大合成DNA（LS-DNA）分子。作为有用的应用，本发明允许以降低的成本和精力从组合的单链DNA片段装配编码全蛋白的基因（通常为1000-3000个核苷酸对）。

9. 发明授权

US08614072B2 Polymerase incorporation of non-standard nucleotides 有权
标题翻译：聚合酶掺入非标准核苷酸
公开(公告)号：US08614072B2
公开(公告)日：2013-12-24
申请号：US12999138
申请日：2009-06-16
申请人： Steven Albert Benner , Zunyi Yang
发明人： Steven Albert Benner , Zunyi Yang
IPC分类号： C12P19/34 , C12Q1/68
CPC分类号： C12Q1/6853 , C12P19/34 , C12Q1/686 , C12Q2521/101 , C12Q2525/117 , C12Q2525/191
摘要： The disclosed invention teaches processes to amplify oligonucleotides by contacting templates and primers with DNA polymerases and triphosphates of non-standard nucleotides, which form nucleobase pairs fitting the standard Watson-Crick geometry, but joined by hydrogen bonding patterns different from those that join standard A:T and G:C pairs. Thus, this invention relates to nucleotide analogs and their derivatives that, when incorporated into DNA and RNA, expand the number of replicatable nucleotides beyond the four found in standard DNA and RNA. The invention further relates to polymerases that incorporate those non-standard nucleotide analogs into oligonucleotide products using the corresponding triphosphate derivatives, and more specifically, polymerases and non-standard nucleoside triphosphates that support the polymerase chain reaction (PCR), including PCR where the products contain more than one non-standard nucleotide unit.
摘要翻译：所公开的发明教导了通过使模板和引物与DNA聚合酶和非标准核苷酸的三磷酸接触来扩增寡核苷酸的过程，其形成适合标准Watson-Crick几何形状的核碱基对，但是通过与加入标准A： T和G：C对。因此，本发明涉及核苷酸类似物及其衍生物，其在掺入DNA和RNA时扩大超出标准DNA和RNA中发现的四种可重复核苷酸的数量。本发明还涉及使用相应的三磷酸衍生物，更具体地，支持聚合酶链式反应（PCR）的聚合酶和非标准核苷三磷酸（包括产物含有的PCR）的寡核苷酸产物，将这些非标准核苷酸类似物结合到聚合酶中的聚合酶多个非标准核苷酸单位。

10. 发明授权

US08586303B1 In vitro selection with expanded genetic alphabets 有权
标题翻译：用扩增的基因字母表进行体外选择
公开(公告)号：US08586303B1
公开(公告)日：2013-11-19
申请号：US13493172
申请日：2012-06-11
申请人： Steven Albert Benner , Zunyi Yang
发明人： Steven Albert Benner , Zunyi Yang
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6811 , C12Q2521/345 , C12Q2525/117 , C12Q2525/205
摘要： This invention enables processes for extracting from a mixture of oligonucleotide molecules individuals that bind to a preselected target (aptamers) or catalyze a preselected reaction (xNAzymes) that contain one or more non-standard nucleotides. These pair with their complements in a Watson-Crick geometry with a pattern of hydrogen bonds different from that pairing adenine and thymine, and guanine and cytosine. The processes comprise (a) obtaining this mixture containing non-standard nucleotides within preselected regions, (b) contacting the mixture with a preselected target or one or more reactants for the reaction whose catalysis is desired, (c) separating oligonucleotides having a greater affinity to the target or catalytic effectiveness from the remainder that have less affinity or catalytic effectiveness, and (d) amplifying the separated oligonucleotides. The process exploits PCR conditions that amplify oligonucleotides with less than 5% loss of the non-standard nucleotide per cycle, and processes to determine the sequences of the amplified oligonucleotides.
摘要翻译：本发明使得能够从寡核苷酸分子的混合物提取与结合预选靶（适体）的个体或催化包含一个或多个非标准核苷酸的预选反应（xNAzymes）的方法。这些与他们在沃森 - 克里克几何中的补体配对，具有与该配对腺嘌呤和胸腺嘧啶不同的氢键模式，以及鸟嘌呤和胞嘧啶。所述方法包括（a）获得在预选区域内含有非标准核苷酸的混合物，（b）使混合物与预选靶物或一种或多种反应物接触，用于需要其催化的反应，（c）分离具有较大亲和力的寡核苷酸与具有较小亲和性或催化效力的其余部分的靶标或催化有效性，和（d）扩增分离的寡核苷酸。该过程利用扩增每个周期中非标准核苷酸少于5％的寡核苷酸的PCR条件，以及确定扩增的寡核苷酸序列的方法。

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