Improved methods of determining nucleic acid structural information

A technology for nucleic acid and nucleic acid molecules, applied in the field of nano, genome assembly and sample analysis based on single-molecule visualization and analysis

Active Publication Date: 2016-10-12
BIONANO GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This presents a further challenge to lower sequencing costs and defeats the original primary goal of significantly reducing sequencing costs below the target $1,000 boundary

Method used

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  • Improved methods of determining nucleic acid structural information
  • Improved methods of determining nucleic acid structural information
  • Improved methods of determining nucleic acid structural information

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1-Labeling with fluorescent SAM

[0131] The human DNA containing megabases is treated with a methyltransferase (MTase) selected from Table 1 in the presence of a modified S-adenosylmethionine (SAM) containing a fluorophore or multiple fluorophores. After covalently transferring the fluorophore-SAM complex to the methyltransferase target site, the labeled DNA was stained with yoyo I for processing on the Irys system (BioNanoGenomics). Briefly, the DNA is linearized in the overall parallel nanochannel to achieve ~80% stretch, excited with a laser suitable for the detection of the skeleton and the marker, and optical imaging is performed to show the pattern of the marker on the DNA molecule . Adjust MTase labeling conditions to achieve reference genome> 40% of the drawing and <20% error rate. Query molecules ≥ 150Kb to overlap and map to generate a genome map.

Embodiment 2

[0132] Example 2-Mark with click (alkynes-azide-Cu-ligand)

[0133] The human DNA containing megabases was treated with a methyltransferase selected from Table 1 in the presence of a modified S-adenosylmethionine (SAM) containing an alkyne transfer moiety. After the alkyne group is covalently transferred to the methyltransferase target site, a copper-catalyzed coupling reaction is used to fluorescently label the alkyne label site in the presence of a copper coordination ligand (such as BTTAA, BTTES), To prevent copper-induced DNA fragmentation. As described in Example 1, the labeled DNA was stained with yoyo I for processing on the Irys system.

Embodiment 3

[0134] Example 3-Mark with a click (azide-DBCO)

[0135] The human DNA containing megabases was treated with a methyltransferase selected from Table 1 in the presence of a modified S-adenosylmethionine (SAM) containing an azide transfer moiety. After covalently transferring the azide group to the methyltransferase target site, a DBCO-fluorophore conjugate is used to fluorescently label the azide labeling site (copper-free click chemistry). As described in Example 1, the labeled DNA was stained with yoyo I for processing on the Irys system. Examples of DBCO-fluorophore conjugates include coupling DBCO directly to a fluorophore, or coupling DBCO to a moiety that has been coupled to multiple fluorophores (for example, a DBCO oligomer containing multiple fluorophores) Or dendrimer).

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Abstract

Methods of double-stranded nucleic acid sequence determination and assembly that are able to identify insertions, deletions, repeat region sizes and genomic rearrangements, for example, are disclosed herein, which can use relatively large labeled nucleic acid fragments to analyze the structure of even larger genetic regions. In some embodiments these methods involve the use of certain parameters which unexpectedly improve overall method performance. In some embodiments these methods involve sample labeling that does not result in the formation of single-stranded nucleic acid fragment labeling intermediaries.

Description

[0001] Related application [0002] This application is based on 35 U.S.C. §119(e) requesting priority of U.S. Provisional Application No. 61 / 941,261 filed on February 18, 2014, the content of which is expressly incorporated herein by reference in its entirety. Technical field [0003] The present invention relates to the field of nanotechnology, and the field of genome assembly and sample analysis based on single molecule visualization and analysis. Background technique [0004] Macromolecules such as DNA or RNA are long polymer chains composed of nucleotides, and their linear sequence is directly related to the gene expression information of the source organism's genome and post-genome. Sequence regions, motifs, and functional units such as open reading frames (ORF), untranslated regions (UTR), exons, introns, protein factor binding sites, epigenomic sites such as CpG clusters, microRNA sites The direct sequencing and mapping of transposons, retrotransposons and other structural ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G16B20/30
CPCC12Q1/6869G16B20/30C12Q2521/125C12Q2563/107C12Q2539/113G16B20/00C12Q2563/155C12Q2565/629
Inventor 米夏埃尔·G·萨格比尼亨利·B·萨多夫斯基戈兰·普列瓦利契奇亚历克斯·R·黑斯蒂曹涵
Owner BIONANO GENOMICS
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