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Analysis of polynucleotides

A polynucleotide and nucleic acid technology, applied in the field of nanotechnology and nucleic acid analysis, can solve the problems of inability to directly quantify the copy number and low accuracy

Active Publication Date: 2016-06-15
BIONANO GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many types of genome-wide approaches exist for detecting disease, many also cannot directly quantify specific copy numbers of pathogenic polynucleotides within the host genome, map the position of pathogenic nucleotides within the host genome, and assemble large complex Genomes to locate extragenomic DNA material, where current techniques and methods have very low accuracy

Method used

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  • Analysis of polynucleotides
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Examples

Experimental program
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Effect test

Embodiment 1

[0108] Example 1: De novo generation of genome maps

[0109] De novo assembly of the human genome map, using automation Nanochannel chips and an automated imaging system purchased from BionanoGenomics, San Diego, California, were used to extract genomic DNA from human cell lines and perform site-specific labeling, followed by linearization and analysis in nanochannels at 50x coverage, where the throughput Running (3-4hrs) for 5-10Gb / chip and further 50-200Gb / chip in the future. To detect site-specific markers and determine distances between markers, raw molecules (>20kb) were used in pairwise pattern matching and de novo assembly processes. image 3 The assembled human genome map is shown, indicating the ease of generating genome maps from large complex genomes. Thus, genome maps can be generated from large complex genomes using the methods described herein.

Embodiment 2

[0110] Example 2: Identification of Structural Changes Compared to a Reference Sequence

[0111] In the assembled genome of Example 1, hundreds of small and large structural variants were detected. An exemplary tandem repeat on chromosome 5 is as Figure 4 shown. Based on the site-specific marker patterns, the genomic sequence was assembled and compared to the pattern of a reference genomic sequence32. A tandem duplication of genomic region 34 comprising an additional 3 tandem copies was identified. From this, de novo genome assembly and structural variant analysis based on site-specific marker patterns can be performed.

Embodiment 3

[0112] Example 3: Preparation of labeled DNA

[0113] DNA was isolated using the plug lysis method. Those skilled in the art will recognize that suitable isolated DNA samples can be prepared using a variety of techniques well known in the art in addition to the exemplary procedures provided herein. Epstein-Barr virus (EBV) infected cells were grown to log phase and harvested. Prepare 2% agarose and water bath by immersing 2% agarose in boiling water for 10-15min until completely melted, put the agarose in 43°C water bath for at least 30min before use, warm the 50ml tube attached to THERMOMIXER to 50°C , and add 10-20ml of water to simulate a water bath. By placing 4x10 6 Cells were prepared by aspirating into a 15 ml tube and centrifuging at 2,200 rpm (1,000 rcf) for 5 minutes. Cells were washed twice in 1XPBS, suspended in 750 μl suspension buffer, and warmed to 43°C. 250 μl of melted agarose was added to the cell suspension and mixed, filling each well in a volume of ...

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Abstract

According to some embodiments herein, methods and kits for labeling and analyzing nucleic acids are provided, In some embodiments, sequence-specific labeling is performed on polynucleotide sequences associated with a host genome, and the presence or absence of patterns characteristic of extragenomic sequences are determined.

Description

[0001] cross application [0002] This application claims priority to US Provisional Patent Application No. 61 / 833,378, filed June 10, 2013, entitled "Polynucleotide Analysis," which is incorporated herein by reference in its entirety. Background technique [0003] A polynucleotide, such as DNA or RNA, is a long polymeric chain of nucleotides whose linear sequence is directly related to the genomic and post-genomic gene expression information of the host organism. A polynucleotide can be single-stranded or double-stranded. [0004] For sequence regions, motifs and functional units such as open reading frames (ORFs), untranslated regions (UTRs), exons, introns, protein factor binding sites, epigenetic sites such as CpG clusters, microRNA sites Direct sequencing and mapping of transposons, retrotransposons, and other structural and functional units can be used to assess genome composition, detect the presence of extragenomic polynucleotides in biological samples, and assess the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C07H21/00C07H21/02G16B30/10
CPCC12Q1/6858G16B30/00G16B30/10C12Q2537/16C12Q2561/109C12Q2565/101C12Q1/705C12Q1/6886C12Q1/6893Y02A50/30
Inventor 曹涵亚历克斯·R·海斯蒂恩内斯特·特兹-特森·拉姆泽利科·扎库拉
Owner BIONANO GENOMICS
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