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1. 发明申请

US20090062129A1 REAGENTS, METHODS, AND LIBRARIES FOR GEL-FREE BEAD-BASED SEQUENCING 审中-公开
标题翻译：用于无凝胶基序列测序的试剂，方法和图谱
公开(公告)号：US20090062129A1
公开(公告)日：2009-03-05
申请号：US11737308
申请日：2007-04-19
申请人： Kevin McKernan , Alan Blanchard , Gina Costa
发明人： Kevin McKernan , Alan Blanchard , Gina Costa
IPC分类号： C40B20/02 , C40B50/06 , C40B40/08
CPC分类号： C12Q1/6837 , C12Q1/6869 , C12Q1/6874 , C12Q2565/501 , C12Q2533/107 , C12Q2531/137 , C12Q2537/1373 , C12Q2535/00
摘要： The present disclosure provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles typically comprise steps of extension, ligation, and cleavage. In certain embodiments, the methods make use of extension probes containing phosphorothiolate linkages and agents capable of cleaving such linkages. Methods of determining information about a sequence using at least two distinguishably labeled probe families are provided, as are methods of performing multiple sequencing reactions on a single template. Automated sequencing systems, flow cells, image processing methods, and computer-readable media that store computer-executable instructions and/or sequence information that can be used in accordance with such methods are also provided. In certain embodiments, blocking oligonucleotides are provided to facilitate sequencing using disclosed methods.
摘要翻译：本公开提供了通过沿单链模板进行连续循环双链延伸来确定核酸序列的方法。循环通常包括延伸，连接和切割的步骤。在某些实施方案中，所述方法利用含有硫代硫醇键的延伸探针和能够切割这种键的试剂。提供了使用至少两个可区分标记的探针家族确定序列信息的方法，以及在单个模板上进行多个测序反应的方法。还提供了自动排序系统，流动池，图像处理方法和存储可根据这些方法使用的计算机可执行指令和/或序列信息的计算机可读介质。在某些实施方案中，提供了阻断寡核苷酸以便于使用公开的方法测序。

2. 发明申请

US20090181385A1 Reagents, methods, and libraries for bead-based sequencing 审中-公开
公开(公告)号：US20090181385A1
公开(公告)日：2009-07-16
申请号：US12220201
申请日：2008-07-21
申请人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
发明人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6874 , B82Y15/00 , B82Y30/00 , C12Q1/68 , C12Q1/6837 , C12Q1/6844 , C12Q1/6869 , C12Q2565/537 , C12Q2565/518 , C12Q2565/513 , C12Q2565/137 , C12Q2565/102 , C12Q2533/107 , C12Q2537/165
摘要： The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled probes containing phosphorothiolate linkages. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.

3. 发明申请

US20090181860A1 Reagents, methods, and libraries for bead-based sequencing 有权
标题翻译：用于珠基测序的试剂，方法和文库
公开(公告)号：US20090181860A1
公开(公告)日：2009-07-16
申请号：US12220208
申请日：2008-07-21
申请人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
发明人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
IPC分类号： C40B40/06 , C07H21/04
CPC分类号： C12Q1/6874 , B82Y15/00 , B82Y30/00 , C12Q1/68 , C12Q1/6837 , C12Q1/6844 , C12Q1/6869 , C12Q2565/537 , C12Q2565/518 , C12Q2565/513 , C12Q2565/137 , C12Q2565/102 , C12Q2533/107 , C12Q2537/165
摘要： The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled probes containing phosphorothiolate linkages. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.
摘要翻译：本发明提供了通过沿着单链模板进行连续循环的双链延伸来确定核酸序列的方法。循环包括延伸，连接和优选切割的步骤。在某些实施方案中，所述方法利用含有硫代磷酸酯键的延伸探针，并使用适合切割这种连接的试剂。本发明提供使用至少两个可区分标记的探针家族确定关于序列的信息的方法。在某些实施方案中，该方法在每个周期中从模板中的多个核苷酸中的每一个获取少于2位的信息。在某些实施方案中，测序反应在附着于固定化珠粒的模板上进行。本发明还提供了含有硫代磷酸酯键的标记探针的集合。此外，本发明包括通过除去初始化寡核苷酸和延伸的链并使用不同的初始化寡核苷酸进行后续反应，在单个模板上进行多个测序反应。

4. 发明授权

US08431691B2 Reagents, methods, and libraries for bead-based sequencing 有权
公开(公告)号：US08431691B2
公开(公告)日：2013-04-30
申请号：US12220208
申请日：2008-07-21
申请人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
发明人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
IPC分类号： C07H21/04
CPC分类号： C12Q1/6874 , B82Y15/00 , B82Y30/00 , C12Q1/68 , C12Q1/6837 , C12Q1/6844 , C12Q1/6869 , C12Q2565/537 , C12Q2565/518 , C12Q2565/513 , C12Q2565/137 , C12Q2565/102 , C12Q2533/107 , C12Q2537/165
摘要： The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled probes containing phosphorothiolate linkages. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.

5. 发明申请

US20080003571A1 Reagents, methods, and libraries for bead-based sequencing 审中-公开
公开(公告)号：US20080003571A1
公开(公告)日：2008-01-03
申请号：US11345979
申请日：2006-02-01
申请人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
发明人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6874 , B82Y15/00 , B82Y30/00 , C12Q1/68 , C12Q1/6837 , C12Q1/6844 , C12Q1/6869 , C12Q2565/537 , C12Q2565/518 , C12Q2565/513 , C12Q2565/137 , C12Q2565/102 , C12Q2533/107 , C12Q2537/165
摘要： The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. In certain embodiments the methods make use of extension probes containing an abasic residue or a damaged base and employ agents appropriate to cleave linkages between a nucleoside and an abasic residue and/or agents appropriate to remove a damaged base from a nucleic acid. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to beads, which are immobilized in or on a semi-solid support. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages or trigger residues that are suitable for use in the method. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides. The invention further provides efficient methods for preparing templates, particularly for performing sequencing multiple different templates in parallel. The invention also provides methods for performing ligation and cleavage. The invention also provides new libraries of nucleic acid fragments containing paired tags, and methods of preparing microparticles having multiple different templates (e.g., containing paired tags) attached thereto and of sequencing the templates individually. The invention also provides automated sequencing systems, flow cells, image processing methods, and computer-readable media that store computer-executable instructions (e.g., to perform the image-processing methods) and/or sequence information. In certain embodiments the sequence information is stored in a database.

6. 发明授权

US08329404B2 Reagents, methods, and libraries for bead-based sequencing 有权
标题翻译：用于珠基测序的试剂，方法和文库
公开(公告)号：US08329404B2
公开(公告)日：2012-12-11
申请号：US13410919
申请日：2012-03-02
申请人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
发明人： Kevin McKernan , Alan Blanchard , Lev Kotler , Gina Costa
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6874 , B82Y15/00 , B82Y30/00 , C12Q1/68 , C12Q1/6837 , C12Q1/6844 , C12Q1/6869 , C12Q2565/537 , C12Q2565/518 , C12Q2565/513 , C12Q2565/137 , C12Q2565/102 , C12Q2533/107 , C12Q2537/165
摘要： The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.
摘要翻译：本发明提供了通过沿着单链模板进行连续循环的双链延伸来确定核酸序列的方法。循环包括延伸，连接和优选切割的步骤。在某些实施方案中，所述方法利用含有硫代磷酸酯键的延伸探针，并使用适合切割这种连接的试剂。本发明提供使用至少两个可区分标记的探针家族确定关于序列的信息的方法。在某些实施方案中，该方法在每个周期中从模板中的多个核苷酸中的每一个获取少于2位的信息。在某些实施方案中，测序反应在附着于固定化珠粒的模板上进行。本发明还提供了含有硫代磷酸酯键的标记的延伸探针的集合。此外，本发明包括通过除去初始化寡核苷酸和延伸的链并使用不同的初始化寡核苷酸进行后续反应，在单个模板上进行多个测序反应。

7. 发明授权

US07993842B2 Directed enrichment of genomic DNA for high-throughput sequencing 失效
标题翻译：导向富集基因组DNA进行高通量测序
公开(公告)号：US07993842B2
公开(公告)日：2011-08-09
申请号：US12504485
申请日：2009-07-16
申请人： Kevin McKernan , Alan Blanchard , Douglas R. Smith
发明人： Kevin McKernan , Alan Blanchard , Douglas R. Smith
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6846 , C12Q1/6837 , C12Q2565/537 , C12Q2547/107 , C12Q2537/143
摘要： The present invention provides microarrays of oligonucleotide primer pairs, and in particular, microarrays of primers that comprise at least one cleavable linkage. Also provided are methods to capture oligonucleotide primer pairs from one or more microarrays, and methods to use the captured oligonucleotide primer pairs, such as for amplification of a target polynucleotide sequence. In addition, methods of using a microarray to isolate, purify and/or amplify a target polynucleotide are provided.
摘要翻译：本发明提供寡核苷酸引物对的微阵列，特别是包含至少一个可切割键的引物的微阵列。还提供了从一个或多个微阵列捕获寡核苷酸引物对的方法，以及使用捕获的寡核苷酸引物对的方法，例如扩增靶多核苷酸序列。此外，提供了使用微阵列分离，纯化和/或扩增靶多核苷酸的方法。

8. 发明申请

US20070231823A1 Directed enrichment of genomic DNA for high-throughput sequencing 审中-公开
标题翻译：导向富集基因组DNA进行高通量测序
公开(公告)号：US20070231823A1
公开(公告)日：2007-10-04
申请号：US11726719
申请日：2007-03-22
申请人： Kevin McKernan , Alan Blanchard , Douglas Smith
发明人： Kevin McKernan , Alan Blanchard , Douglas Smith
IPC分类号： C12Q1/68 , C12P19/34 , C12M3/00
CPC分类号： C12Q1/6846 , C12Q1/6837 , C12Q2565/537 , C12Q2547/107 , C12Q2537/143
摘要： The present invention provides microarrays of oligonucleotide primer pairs, and in particular, microarrays of primers that comprise at least one cleavable linkage. Also provided are methods to capture oligonucleotide primer pairs from one or more microarrays, and methods to use the captured oligonucleotide primer pairs, such as for amplification of a target polynucleotide sequence. In addition, methods of using a microarray to isolate, purify and/or amplify a target polynucleotide are provided.
摘要翻译：本发明提供寡核苷酸引物对的微阵列，特别是包含至少一个可切割键的引物的微阵列。还提供了从一个或多个微阵列捕获寡核苷酸引物对的方法，以及使用捕获的寡核苷酸引物对的方法，例如扩增靶多核苷酸序列。此外，提供了使用微阵列分离，纯化和/或扩增靶多核苷酸的方法。

9. 发明申请

US20070054285A1 Method for isolating nucleic acids 审中-公开
标题翻译：分离核酸的方法
公开(公告)号：US20070054285A1
公开(公告)日：2007-03-08
申请号：US11399310
申请日：2006-04-06
申请人： Kevin McKernan , Junaid Ziauddin
发明人： Kevin McKernan , Junaid Ziauddin
IPC分类号： C12Q1/68 , C07H21/04
CPC分类号： C12N15/1006 , C12N15/1013
摘要： Described herein is a method in which genomic nucleic acid of a cell can be separated from nucleic acid having a molecular weight that is lower than the molecular weight of the genomic nucleic acid (e.g., plasmid DNA) of the cell directly from a cell growth culture. Also described herein, a method in which genomic nucleic acid can be separated from nucleic acid having a molecular weight that is lower than the molecular weight of the genomic nucleic acid in a cell lysate without the need to prepare a cleared lysate.
摘要翻译：本文描述了一种方法，其中细胞的基因组核酸可从直接来自细胞生长培养物的细胞的分子量低于细胞的基因组核酸（例如质粒DNA）的分子量的核酸分离。本文还描述了可以将基因组核酸与细胞裂解物中分子量低于基因组核酸分子量的核酸分离而不需要制备澄清的裂解物的方法。

10. 发明申请

US20060177836A1 Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers 有权
公开(公告)号：US20060177836A1
公开(公告)日：2006-08-10
申请号：US11194072
申请日：2005-07-29
申请人： Kevin McKernan , Erik Gustafson , Adrianne Brand
发明人： Kevin McKernan , Erik Gustafson , Adrianne Brand
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6806 , C12N15/1006 , C12N15/1013 , C12Q1/6834 , C12Q1/6869 , C12Q2527/125
摘要： The present invention is directed to a method of isolating a target species (e.g., target nucleic acid species) from a mixture. In the methods of the present invention, the mixture is combined with solid phase carriers having a surface comprising multiple functional groups one of which reversibly and selectively binds the target species. In a particular embodiment, the mixture is combined with solid phase carriers having a first functional group which reversibly binds nucleic acids and a second functional group which selectively and reversibly binds the target nucleic acid species, thereby producing a first combination. The first combination is maintained under conditions appropriate for binding of the nucleic acids to the first functional group and binding of the target nucleic acid species to the second functional group. The solid phase carriers are separated from the first combination, and combined with an agent (e.g., buffer) that selectively removes (e.g., elutes) either the nucleic acid from the first functional group or the target nucleic acid species from the second functional group of the solid phase carriers, thereby isolating a target nucleic acid species from a mixture comprising a plurality of nucleic acid species.

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