公开(公告)号：US20130115598A1

公开(公告)日：2013-05-09

申请号：US13652431

申请日：2012-10-15

Applicant: Lawrence Loeb ,  Jiang-Cheng Shen ,  Edward J. Fox

Inventor： Lawrence Loeb ,  Jiang-Cheng Shen ,  Edward J. Fox

IPC: C12Q1/68

CPC classification number: C12Q1/6827 ,  C12N15/1082 ,  C12Q1/6858 ,  C12Q1/6886 ,  C12Q2600/106 ,  C12Q2521/514 ,  C12Q2525/101 ,  C12Q2545/114

Abstract: Methods to measure a variety of DNA synthetic processes in live human cells by introducing and retrieving exogenous DNA probes are provided herein. Using fragments of bacterial plasmid or phage DNA, a wide array of DNA constructs may be assembled to mimic the intermediates of DNA transactions, including replication, translation synthesis, and end-joining. These DNA probes may be transfected into human cells and retrieved for mutational analysis using a modified Random Mutation Capture assay or NextGen DNA sequencing. These assays require only a small number of cells, such as might be available from biopsy material. Thus, the methods described herein may be applied to the early detection of cancer, predicting the responsiveness of individual cancers to chemotherapy, and measuring the DNA repair capacity of individuals to environmental DNA damaging agents. This approach may be automated and used for screening human populations for variations in DNA synthetic and repair activities.

Abstract translation: 本文提供了通过引入和回收外源DNA探针来测量活细胞中各种DNA合成过程的方法。 使用细菌质粒或噬菌体DNA的片段，可以组合各种DNA构建体以模拟DNA事务的中间体，包括复制，翻译合成和末端连接。 可以将这些DNA探针转染到人细胞中并使用修饰的随机突变捕获测定法或NextGen DNA测序法进行突变分析。 这些测定仅需要少量的细胞，例如可以从活检材料获得。 因此，本文描述的方法可以应用于癌症的早期检测，预测个体癌症对化疗的反应性，以及测量个体对环境DNA损伤剂的DNA修复能力。 这种方法可以是自动化的，并用于筛选人类DNA合成和修复活动的变化。

公开(公告)号：US20080045451A1

公开(公告)日：2008-02-21

申请号：US11558246

申请日：2006-11-09

Applicant: Lawrence Loeb ,  Werner Geurtsen ,  Ray Monnat

Inventor： Lawrence Loeb ,  Werner Geurtsen ,  Ray Monnat

IPC: A61K31/70 ,  A61P43/00 ,  C07H21/04 ,  C07K16/00 ,  C12N15/00 ,  C12N5/00

CPC classification number: C12N9/1007 ,  C12Y201/01045

Abstract: Novel thymidylate synthase (TS mutants) are disclosed differing from human wild type thymidylate synthase in single, double, or multiple mutations, which show intact enzyme activity and enhanced resistance to TS-inhibiting drugs like 5-fluorouracil or 5-fluoro-2-deoxyuridinemonophosphate. All these mutants can be used for the protection of normal human cell populations against the toxic manifestation of analogs that inhibited TS. Drugs for the local treatment or prevention of a mucositis in the oral cavity and the gastrointestinal tract caused by chemotherapy with TS-inhibitors are also provided.

Abstract translation: 公开了新的胸苷酸合酶（TS突变体），其在单个，双重或多个突变中与人类野生型胸苷酸合酶不同，其显示完整的酶活性和增强抗TS抑制药物如5-氟尿嘧啶或5-氟-2-脱氧嘌呤二磷酸 。 所有这些突变体可用于保护正常人细胞群体免受抑制TS的类似物的毒性表现。 还提供了用于通过TS抑制剂化疗引起的口腔和胃肠道局部治疗或预防粘膜炎的药物。

公开(公告)号：US20070111242A1

公开(公告)日：2007-05-17

申请号：US11582372

申请日：2006-10-18

Applicant: Dipak Dube ,  Marshall Horwitz ,  Lawrence Loeb

Inventor： Dipak Dube ,  Marshall Horwitz ,  Lawrence Loeb

IPC: C12Q1/68 ,  G06F19/00

CPC classification number: C12N15/66 ,  C12N15/102 ,  C12N15/1058 ,  C12N15/70 ,  C12P19/34

Abstract: A method of obtaining an oligonucleotide capable of carrying out a predetermined biological function. A heterogeneous pool of oligonucleotides, x+y+z nucleotides in length, is first generated. Each oligonucleotide has a 5′ randomized sequence, x nucleotides in length, a central preselected sequence, y nucleotides in length, and a 3′ randomized sequence, z nucleotides in length. The resulting heterogeneous pool contains nucleic acid sequences representing a random sampling of the 4x+z possible sequences for oligonucleotides of the stated length. A random sampling of the heterogeneous pool of oligonucleotides is introduced Into a population of cells that do not exhibit the predetermined biological function. The population of engineered cells Is then screened for a subpopulation of cells exhibiting the predetermined biological function. From that subpopulation of cells Is Isolated an oligonucleotide containing the preselected sequence and capable of carrying out the predetermined biological function.

Abstract translation: 一种获得能够实现预定生物学功能的寡核苷酸的方法。 寡核苷酸长度的x + y + z核苷酸的异质库首先被产生。 每个寡核苷酸具有5'随机序列，长度为x核苷酸，中心预选序列，长度为y个核苷酸，长度为3'的随机序列，z核苷酸。 得到的异源池含有表示所述长度的寡核苷酸的4×x + z +可能序列的随机取样的核酸序列。 将寡核苷酸的异源库的随机抽样引入到不具有预定生物学功能的细胞群中。 然后筛选工程细胞群体，以获得表现出预定生物学功能的细胞亚群。 从细胞的亚群分离出含有预选序列并能够实现预定生物学功能的寡核苷酸。

公开(公告)号：US20050196800A1

公开(公告)日：2005-09-08

申请号：US11096645

申请日：2005-03-31

Applicant: Lawrence Loeb ,  Leroy Hood ,  Motoshi Suzuki

Inventor： Lawrence Loeb ,  Leroy Hood ,  Motoshi Suzuki

IPC: C12N9/10 ,  C12N9/12 ,  C12N9/22 ,  C12P19/34 ,  C12Q1/34 ,  C12Q1/68

CPC classification number: C12N9/1252 ,  C12Q1/6858 ,  C12Q1/6883

Abstract: The present invention provides a method for identifying a thermostable polymerase having altered fidelity. The method consists of generating a random population of polymerase mutants by mutating at least one amino acid residue of a thermostable polymerase and screening the population for one or more active polymerase mutants by genetic selection. For example, the invention provides a method for identifying a thermostable polymerase having altered fidelity by mutating at least one amino acid residue in an active site O-helix of a thermostable polymerase. The invention also provides thermostable polymerases and nucleic acids encoding thermostable polymerases having altered fidelity, for example, high fidelity polymerases and low fidelity polymerases. The invention additionally provides a method for identifying one or more mutations in a gene by amplifying the gene with a high fidelity polymerase. The invention further provides a method for accurately copying repetitive nucleotide sequences using a high fidelity polymerase mutant. The invention also provides a method for diagnosing a genetic disease using a high fidelity polymerase mutant. The invention further provides a method for randomly mutagenizing a gene by amplifying the gene using a low fidelity polymerase mutant.

Abstract translation: 本发明提供了鉴定具有改变的保真度的热稳定聚合酶的方法。 该方法包括通过突变热稳定聚合酶的至少一个氨基酸残基并通过遗传选择筛选一个或多个活性聚合酶突变体的群体来产生聚合酶突变体的随机群体。 例如，本发明提供了通过突变热稳定聚合酶的活性位点O-螺旋中的至少一个氨基酸残基来鉴定具有改变的保真度的热稳定聚合酶的方法。 本发明还提供了具有改变的保真度的热稳定聚合酶和编码耐热聚合酶的核酸，例如高保真聚合酶和低保真度聚合酶。 本发明另外提供了通过用高保真聚合酶扩增基因来鉴定基因中的一个或多个突变的方法。 本发明还提供了使用高保真聚合酶突变体准确地复制重复核苷酸序列的方法。 本发明还提供了使用高保真聚合酶突变体来诊断遗传疾病的方法。 本发明还提供了使用低保真度聚合酶突变体扩增基因来随机诱变基因的方法。

公开(公告)号：US20050187180A1

公开(公告)日：2005-08-25

申请号：US11098796

申请日：2005-04-04

Applicant: Lawrence Loeb ,  James Mullins

Inventor： Lawrence Loeb ,  James Mullins

IPC: A61K39/12 ,  A61K39/21 ,  A61K39/29 ,  A61K45/06 ,  C12N7/04 ,  C12N7/06 ,  C12N15/10 ,  A61K48/00 ,  C12N15/86 ,  C12N7/00

CPC classification number: A61K31/7076 ,  A61K31/7068 ,  A61K31/7072 ,  A61K31/708 ,  A61K39/00 ,  A61K45/06 ,  C12N7/00 ,  C12N15/102 ,  C12N2710/16063 ,  C12N2740/16061 ,  C12N2740/16063 ,  C12N2770/24161 ,  C12N2770/24261 ,  Y02A50/386 ,  Y02A50/464

Abstract: The present invention is directed to the identification and use of ribonucleoside analogs to induce the mutation of an RNA virus, including BVDV, HIV and HCV, or a virus which otherwise replicates through an RNA intermediate. The increase in the mutation rate of the virus results in reduced viability of progeny generations of the virus, thereby inhibiting viral replication. In addition to these methods and related compositions, the invention provides methods and combinatorial chemistry libraries for screening ribonucleoside analogs for mutagenic potential.

公开(公告)号：US20060008806A1

公开(公告)日：2006-01-12

申请号：US10757590

申请日：2004-01-15

Applicant: Dipak Dube ,  Marshall Horwitz ,  Lawrence Loeb

Inventor： Dipak Dube ,  Marshall Horwitz ,  Lawrence Loeb

IPC: C12Q1/68 ,  C12P19/34

CPC classification number: C12N15/66 ,  C12N15/102 ,  C12N15/1058 ,  C12N15/70 ,  C12P19/34

Abstract: A method of obtaining an oligonucleotide capable of carrying out a predetermined biological function. A heterogeneous pool of oligonucleotides, x+y+z nucleotides in length, is first generated. Each oligonucleotide has a 5′ randomized sequence, x nucleotides in length, a central preselected sequence, y nucleotides in length, and a 3′ randomized sequence, z nucleotides in length. The resulting heterogeneous pool contains nucleic acid sequences representing a random sampling of the 4x+z possible sequences for oligonucleotides of the stated length. A random sampling of the heterogeneous pool of oligonucleotides is introduced into a population of cells that do not exhibit the predetermined biological function. The population of engineered cells is then screened for a subpopulation of cells exhibiting the predetermined biological function. From that subpopulation of cells is isolated an oligonucleotide containing the preselected sequence and capable of carrying out the predetermined biological function.

Abstract translation: 一种获得能够实现预定生物学功能的寡核苷酸的方法。 寡核苷酸长度的x + y + z核苷酸的异质库首先被产生。 每个寡核苷酸具有5'随机序列，长度为x核苷酸，中心预选序列，长度为y个核苷酸，长度为3'的随机序列，z核苷酸。 所得到的异源池含有表示所述长度的寡核苷酸的4×x + z +可能序列的随机取样的核酸序列。 将寡核苷酸的异源池的随机抽样引入到不具有预定生物学功能的细胞群中。 然后筛选工程细胞群体以获得表现出预定生物学功能的细胞亚群。 从细胞的亚群分离出含有预选序列并能够实现预定生物学功能的寡核苷酸。