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Method and kit for quickly extracting long-fragment genomic DNA with single reaction tube

A genome and kit technology, applied in the field of rapid single-reaction tube extraction of long fragments of genomic DNA, can solve problems such as complex operations and long time-consuming

Pending Publication Date: 2020-07-07
BERRYGENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problems of long time-consuming and complicated operation during the extraction of existing long-segment genomic DNA

Method used

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  • Method and kit for quickly extracting long-fragment genomic DNA with single reaction tube
  • Method and kit for quickly extracting long-fragment genomic DNA with single reaction tube
  • Method and kit for quickly extracting long-fragment genomic DNA with single reaction tube

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Extracting long fragments of genomic DNA according to the method of the present invention

[0041] Using the method of the present invention, the genomic DNA of leukocytes is extracted according to the following steps:

[0042] (1) Take 0.3x10 6 White blood cells were centrifuged at 2000g for 2 minutes at room temperature, and the supernatant was removed;

[0043] (2) Add 30μL of lysis buffer (100mM NaCl, 10mM Tris-HCl (pH8.0), 25mM EDTA (pH8.0), 0.50% SDS) to resuspend the cells, then add 2μL of 2mg / ml proteinase K to mix;

[0044] (3) Place in a metal bath and treat at 50°C for 1 hour;

[0045] (4) Then add 12μL of 5M ammonium acetate and 90μL of absolute ethanol, invert 10 times, place in a shaker at room temperature 20rpm and mix slowly for 5-10 minutes, so that the DNA can completely precipitate and form small clusters;

[0046] (5) Wash DNA twice with 200μL 70% ethanol;

[0047] (6) Dissolve DNA with 42 μL TE buffer (10 mmol / L Tris-HCl (pH 8.0); 1 mmol / L EDTA (pH 8...

Embodiment 2

[0048] Example 2. Extraction of long fragments of genomic DNA according to the method of embedding in low melting point agarose

[0049] Using the traditional method of embedding with low melting point agarose, extract the genomic DNA of leukocytes according to the following steps:

[0050] Step 1: Prepare agarose gel block

[0051] (1) Take 0.3x10 6 Centrifuge at 2000g for 2 minutes to remove the supernatant.

[0052] (2) Resuspend the cells with 65 μL of PBS and equilibrate in a metal bath at 43°C for 10 minutes.

[0053] (3) Add 39 μL of 2% Agarose to mix, and add to the glue hole.

[0054] (4) Place the gel block in a refrigerator at 4°C for 45 minutes to gel.

[0055] Step 2: Digesting the gel block

[0056] (5) Add 2.5 mL of proteinase K solution to a 50 mL centrifuge tube. Transfer the gel block to a 50mL centrifuge tube and place it in a thermostatic mixer at 50°C and mix for 2 hours.

[0057] (6) Add a mesh cover, pour out the proteinase K solution, replace with a new proteinase K...

Embodiment 3

[0067] Example 3. Quality detection of long genomic DNA

[0068] The long-segment genomic DNA obtained in Examples 1 and 2 was subjected to pulse field electrophoresis to test its quality, the results are as follows figure 1 Shown. These results indicate that the method of the present invention can effectively obtain long fragments of genomic DNA with an average length of more than 200 kb and up to Mb level.

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Abstract

The invention relates to a method for quickly extracting long-fragment genomic DNA with a single reaction tube. The method comprises the following steps: a) adding a lysis solution and proteinase K into a sample to lyse cells and release genomic DNA; b) adding a precipitant into a reaction system of the step a) to obtain a precipitate of the genomic DNA; c) washing the obtained precipitate of thegenomic DNA with a cleaning solution; and d) dissolving the genomic DNA with a dissolving solution. The invention also relates to a kit suitable for the method.

Description

Technical field [0001] The invention relates to the field of extracting genomic DNA. Specifically, the present invention relates to a method for extracting long fragments of genomic DNA from a rapid single reaction tube, and a kit suitable for this method. Background technique [0002] The preparation of long-segment genomic DNA molecules has a wide range of needs in the field of biology, especially DNA sequencing and genome assembly. Pacific Biosicences' SMRT long-read sequencing technology can reach 60-100Kb for single-molecule sequencing 1 , While Oxford Nanopore Technology’s nanopore sequencing technology can detect single-molecule DNA fragments up to Mb 2 . 10x Genomics for haplotype splicing and genome assembly 3 And CPT-seq 4 For linked-reads technology, it is necessary to prepare genomic molecular fragments of tens to hundreds of kilobytes in length. BioNano Genomics’ single-molecule optical mapping technology based on the Saphyr platform can assist genome assembly, det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806
Inventor 毛爱平张海满张建光
Owner BERRYGENOMICS CO LTD
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