公开(公告)号：US11047006B2

公开(公告)日：2021-06-29

申请号：US16908611

申请日：2020-06-22

Applicant: UNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION

Inventor： Jesse Salk ,  Lawrence A. Loeb ,  Michael Schmitt

IPC: C12Q1/68 ,  C12Q1/6876 ,  C12Q1/6806 ,  C12Q1/6869

Abstract: Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands.

公开(公告)号：US20190352714A1

公开(公告)日：2019-11-21

申请号：US16503398

申请日：2019-07-03

Applicant: UNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION

Inventor： Jesse Salk ,  Lawrence A. Loeb ,  Michael Schmitt

IPC: C12Q1/6876 ,  C12Q1/6806 ,  C12Q1/6869

Abstract: Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands.

公开(公告)号：US10370713B2

公开(公告)日：2019-08-06

申请号：US16120091

申请日：2018-08-31

Applicant: UNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION

Inventor： Jesse Salk ,  Lawrence A. Loeb ,  Michael Schmitt

IPC: C12Q1/68 ,  C12Q1/6876 ,  C12Q1/6869 ,  C12Q1/6806

Abstract: Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands.

公开(公告)号：US20140051072A1

公开(公告)日：2014-02-20

申请号：US13797207

申请日：2013-03-12

Applicant: University of Washington Through its Center for Commercialization

Inventor： Lawrence A. Loeb ,  Leroy Hood ,  Mitoshi Suzuki

IPC: C12N9/12 ,  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。