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    Methods for Amplification of Nucleic Acids on Solid Support
    1.
    发明申请
    Methods for Amplification of Nucleic Acids on Solid Support 审中-公开
    在固体支持物上扩增核酸的方法

    公开(公告)号：US20160017392A1

    公开(公告)日：2016-01-21

    申请号：US14773362

    申请日：2014-03-14

    申请人： Lyle J. ARNOLD ,  Norman C. NELSON

    发明人： Lyle J. Arnold ,  Norman C. Nelson

    IPC分类号： C12P19/34

    CPC分类号： C12P19/34 ,  C12Q1/6853 ,  C12Q1/6865 ,  C12Q2525/186 ,  C12Q2533/101 ,  C12Q2565/515 ,  C12Q2565/537 ,  C12Q2531/143

    摘要： The present invention provides methods for amplifying a nucleic acid from a sample containing a mixture of nucleic acids utilizing a solid support. Methods are provided utilizing user-defined primer oligonucleotides for directional amplification that assists in further manipulation of the target nucleic acid, such as sequencing. Methods are also provided utilizing blocker and displacer oligonucleotides for generating amplified target nucleic acids of defined length. One of these methods provides a first oligonucleotide and a second oligonucleotide affixed to a solid support or separate solid supports. The first oligonucleotide is blocked to prevent extension from the 3′-terminus and has a sequence complementary to a first portion of a target nucleic acid. The second oligonucleotide has a sequence that is identical to a second portion of the target nucleic acid. In this method, a sample is applied to the solid support and the target nucleic acid within the sample binds said first oligonucleotide. The solid support is then washed to remove unbound nucleic acids. A primer sequence containing a target binding region and a polymerase promoter sequence is then annealed to the bound target nucleic acid and extended producing a first duplex nucleic acid. The target sequence is then removed leaving a first nucleic acid that can now bind the second oligonucleotide. The second oligonucleotide is extended to produce a second duplex nucleic acid that contains a second nucleic acid. The second nucleic acid is then amplified by adding a polymerase.

    摘要翻译： 本发明提供了使用固体支持物从含有核酸混合物的样品中扩增核酸的方法。 使用用于定向扩增的用户定义的引物寡核苷酸提供了有助于进一步操纵靶核酸的方法，例如测序。 还提供了利用用于产生具有一定长度的扩增靶核酸的阻断剂和置换寡核苷酸的方法。 这些方法之一提供了固定在固体支持物上或第一个固体支持物上的第一寡核苷酸和第二寡核苷酸。 封闭第一寡核苷酸以防止从3'末端延伸并具有与靶核酸的第一部分互补的序列。 第二寡核苷酸具有与靶核酸的第二部分相同的序列。 在该方法中，将样品施加到固体支持物上，并且样品内的靶核酸结合所述第一寡核苷酸。 然后洗涤固体支持物以除去未结合的核酸。 然后将含有靶结合区和聚合酶启动子序列的引物序列退火至结合的靶核酸，延伸产生第一双链体核酸。 然后除去靶序列，留下现在可以结合第二寡核苷酸的第一核酸。 扩展第二寡核苷酸以产生含有第二核酸的第二双链体核酸。 然后通过加入聚合酶扩增第二个核酸。

    Methods for Amplifying Fragmented Target Nucleic Acids Utilizing an Assembler Sequence
    3.
    发明申请
    Methods for Amplifying Fragmented Target Nucleic Acids Utilizing an Assembler Sequence 审中-公开
    使用汇编序列扩增分离的靶核酸的方法

    公开(公告)号：US20160068900A1

    公开(公告)日：2016-03-10

    申请号：US14773366

    申请日：2014-03-15

    申请人： Lyle J. ARNOLD ,  Norman C. NELSON

    发明人： Lyle J. Arnold ,  Norman C. Nelson

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/6858 ,  C12Q1/6806 ,  C12Q1/6844 ,  C12Q2525/161 ,  C12Q2525/186 ,  C12Q2525/204 ,  C12Q2531/101

    摘要： The present invention provides methods of amplifying a fragmented target nucleic acid containing short target nucleic acid fragments utilizing an assembler sequence to convert these short fragments into longer sequences enabling their identification and interrogation. This is particularly important when attempting to identify small genetic variations, such as SNVs, present in highly fragmented nucleic acid samples. Amplification is accomplished by hybridizing the short target nucleic acid sequences to the assembler sequence, where these short sequences serve as primers for extension. Since the fragmented target nucleic acids that contain SNVs are utilized as primers on the assembler sequence they are preserved during amplification and can be detected.

    摘要翻译： 本发明提供了利用汇编序列扩增含有短靶核酸片段的片段化靶核酸的方法，以将这些短片段转化成更长的序列，使其能够进行鉴定和询问。 当试图鉴定存在于高度片段化的核酸样品中的小的遗传变异（例如SNV）时，这尤其重要。 通过将短目标核酸序列杂交到汇编序列来完成扩增，其中这些短序列用作扩增的引物。 由于含有SNV的片段化靶核酸作为汇编序列上的引物，因此在扩增过程中被保留并可以被检测。

    Methods for amplification of nucleic acids utilizing clamp oligonucleotides
    6.
    发明授权
    Methods for amplification of nucleic acids utilizing clamp oligonucleotides 有权

    公开(公告)号：US10202629B2

    公开(公告)日：2019-02-12

    申请号：US14773365

    申请日：2014-03-14

    申请人： Lyle J. Arnold ,  Norman C. Nelson

    发明人： Lyle J. Arnold

    IPC分类号： C12P19/34 ,  C12Q1/68 ,  C12Q1/6844

    摘要： The present invention provides methods of amplifying a target nucleic acid utilizing a clamp oligonucleotide comprising a first target-binding region on the 3′-terminus and a second target-binding region on the 5′-terminus and tether region in between. The tether region may comprise a variety of user-defined sequences or elements that allow for further manipulation of the target nucleic acid. Such as, for example, capture followed by amplification, identification and/or sequencing. The target-binding regions bind to the target nucleic acid, the 3′-terminus functions as a primer to initiate extension across the target nucleic acid sequence and ligation of the gap results in formation of a circularized nucleic acid. This circular template can be used in a variety of processes, including amplification and sequencing.

    Methods for amplification of nucleic acids on solid support
    8.
    发明授权
    Methods for amplification of nucleic acids on solid support 有权

    公开(公告)号：US10174352B2

    公开(公告)日：2019-01-08

    申请号：US14773362

    申请日：2014-03-14

    申请人： Lyle J. Arnold ,  Norman C. Nelson

    发明人： Lyle J. Arnold ,  Norman C. Nelson

    IPC分类号： C12Q1/68 ,  C12P19/34 ,  C12Q1/6853 ,  C12Q1/6865 ,  C07H21/02

    摘要： The present invention provides methods for amplifying a nucleic acid from a sample containing a mixture of nucleic acids utilizing a solid support. Methods are provided utilizing user-defined primer oligonucleotides for directional amplification that assists in further manipulation of the target nucleic acid, such as sequencing. Methods are also provided utilizing blocker and displacer oligonucleotides for generating amplified target nucleic acids of defined length. One of these methods provides a first oligonucleotide and a second oligonucleotide affixed to a solid support or separate solid supports. The first oligonucleotide is blocked to prevent extension from the 3′-terminus and has a sequence complementary to a first portion of a target nucleic acid. The second oligonucleotide has a sequence that is identical to a second portion of the target nucleic acid. In this method, a sample is applied to the solid support and the target nucleic acid within the sample binds said first oligonucleotide. The solid support is then washed to remove unbound nucleic acids. A primer sequence containing a target binding region and a polymerase promoter sequence is then annealed to the bound target nucleic acid and extended producing a first duplex nucleic acid. The target sequence is then removed leaving a first nucleic acid that can now bind the second oligonucleotide. The second oligonucleotide is extended to produce a second duplex nucleic acid that contains a second nucleic acid. The second nucleic acid is then amplified by adding a polymerase.