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Method for performing 3' end biotin labeling on DNA fragments

A technology of biotin labeling and fragmentation, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of low labeling efficiency and achieve the effects of wide application range, reduced sample loss and preference, and high labeling efficiency

Active Publication Date: 2013-08-21
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the labeling efficiency is low due to the uncertainty of the terminal nucleotides of the DNA fragments

Method used

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  • Method for performing 3' end biotin labeling on DNA fragments
  • Method for performing 3' end biotin labeling on DNA fragments
  • Method for performing 3' end biotin labeling on DNA fragments

Examples

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preparation example Construction

[0058] Preparation method of PBS buffer solution: dissolve NaCl8g, KCl0.2g, KH in 800ml distilled water 2 PO 4 0.24g, Na 2 HPO 4 12H 2 O3.63g, adjust the pH value to 7.4 with HCl, add water to 1L, 1.034×10 5 Pa high-pressure steam sterilization for 20min, stored at room temperature.

[0059] The cell lysate (pH8.0) is composed of solvent and solute, the solvent is water, the solute and its concentration are: 10mM Tris-HCl, 10mM NaCl, 0.2% (volume ratio) Triton X-100 and 1× protease inhibitor.

[0060] The cell nucleus lysate (pH8.0) is composed of solvent and solute, the solvent is water, the solute and its concentration are: 50mM Tris-HCl, 10mM EDTA, 1g / 100mL SDS and 1× protease inhibitor.

[0061] 2×BW buffer (pH8.0) is composed of solvent and solute, the solvent is water, the solute and its concentration are: 10mM Tris-HCl, 1.0mM EDTA and 2.0M NaCl.

[0062] TWB buffer is composed of solvent and solute, the solvent is water, the solute and its concentration are: 5mM T...

Embodiment 1

[0063] Embodiment 1, preparation the DNA fragment that ultrasonic breaks up

[0064] 1. Formaldehyde cross-linked chromatin and harvested nuclei

[0065] 1. In order to imitate the DNA fragments produced by chromatin conformation capture technology as much as possible, MCF-7 cells were fixed with 1% formaldehyde and reacted at room temperature for 10 minutes.

[0066] 2. Discard the supernatant, add 10ml of PBS buffer solution containing 0.125M glycine, and react at room temperature for 10 minutes.

[0067] 3. Discard the supernatant, add 1ml 0.25% trypsin solution, incubate at 37°C for 10min, then add 0.4ml serum and 10μl protease inhibitor to stop the reaction.

[0068] 4. Scrape the cells and wash with PBS buffer.

[0069] 5. Add 40ml cell lysate and 100μl protease inhibitor, mix thoroughly, and incubate at 4°C for 10-30min.

[0070] 6. Centrifuge at 4°C and 1500g for 5 minutes to collect the precipitate, which is the nucleus with chromatin cross-linked and fixed by form...

Embodiment 2

[0076] Embodiment 2, the DNA fragment of preparation enzyme cut

[0077] 1. Extract the genomic DNA of MCF-7 cells,

[0078] 2. Digest the genomic DNA obtained in step 1 with restriction endonucleases, and recover DNA fragments of 100-1500 bp.

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Abstract

The invention discloses a method for performing 3' end biotin labeling on DNA fragments obtained through ultrasonication. The method comprises the following steps of: I, adding biotin-labeled dCTP while treating the DNA fragments by Klenow fragments and / or T4DNA polymerase, and then filling in; II, adding biotin-labeled dATP while treating DNA fragments by Klenow fragments and / or T4DNA polymerase, and then filling in; III, adding biotin-labeled dGTP while treating DNA fragments by Klenow fragments and / or T4DNA polymerase, and then filling in; and IV, adding biotin-labeled dTTP while treating DNA fragments by Klenow fragments and / or T4DNA polymerase, and then filling in. The method has the advantages that the labeling efficiency is high; the application range is wide; and the steps of the method are few, and thus the method is simple, convenient and feasible.

Description

technical field [0001] The invention relates to a method for biotin-labeling the 3' end of a DNA fragment. Background technique [0002] DNA labeling is a common operation in modern molecular biology experiments. Correspondingly, people have developed many methods to achieve this goal, such as PCR, end replacement, nick translation and so on. However, non-radioactive and blunt-ended methods specifically for labeling the ends of DNA fragments are few and far between. With the development of genome science and the application of high-throughput sequencing technology, especially the rapid development of genome-wide chromatin conformation capture methods, there is an urgent need for simple and efficient DNA fragment end-labeling methods. [0003] Hi-C is a genome-wide chromatin conformation capture technology, in which Job dekker et al. (Belton JM, 2012; Lieberman-Aiden E, 2009) cut chromatin with the restriction endonuclease HindIII, Then use Klenow fragments to perform end-...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 师明磊赵志虎王东王洋
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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