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DNA ligation method and application thereof

A technology of DNA ligase and ligation products, which is applied in the field of DNA detection, can solve problems such as loss and distortion of DNA fragment ratio, and achieve the effect of less self-ligation, high ligation efficiency, and good ligation direction performance

Active Publication Date: 2021-10-08
XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This will lead to distortion or loss of the proportion of different DNA fragments in the library

Method used

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  • DNA ligation method and application thereof
  • DNA ligation method and application thereof
  • DNA ligation method and application thereof

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Embodiment 1

[0059] Embodiment 1, DNA ligation method and DNA quantitative application

[0060] This embodiment provides a DNA ligation method and DNA quantitative application, which mainly includes the following steps:

[0061] Step 1: DNA Digestion

[0062] 1. Add the following reagents to the 200ul PCR tube:

[0063] Reagent name volume DNA endonuclease 1μl 10x buffer 1μl dna 8μl

[0064] The DNA endonuclease is Fragmentase (the inventor also used Tn5 transposase, DNase I or Endonuclease V in the research process, compared with Fragmentase, the experimental effect has no significant difference).

[0065] 2. Gently inhale and mix with the pipette tip, 37°C for 10 minutes.

[0066] Step 2: DNA Ligation

[0067] 1. Add the following reagents to the 200ul PCR tube:

[0068] Reagent name volume concentration range DNA ligase 1μl .0.1U / μl 10x buffer 1μl Synthetic DNA Fragment 1 1μl 0.1μM Synthetic DNA Frag...

Embodiment 2

[0087] Embodiment 2, DNA ligation method and DNA library construction

[0088] This embodiment provides a DNA ligation method and DNA library construction application, which mainly includes the following steps:

[0089] Step 1: DNA Digestion

[0090] 1. Add the following reagents to the 200ul PCR tube:

[0091] Reagent name volume DNA endonuclease 1μl 10x buffer 1μl dna 8μl

[0092] The optional DNA endonuclease is Endonuclease V (the inventor also used Tn5 transposase, DNaseI or Fragmentase in the research process, compared with Endonuclease V, the experimental effect has no significant difference).

[0093] 2. Gently inhale and mix with the pipette tip, 37°C for 10 minutes.

[0094] Step 2: DNA Ligation

[0095] 1. Add the following reagents to the 200ul PCR tube:

[0096] Reagent name volume concentration range DNA ligase 1μl 0.1U / μl 10x buffer 1μl Synthetic DNA Fragment 1 1μl 0.1μM Synt...

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Abstract

The invention provides a DNA ligation method and application thereof. According to the present invention, firstly, fragments for DNA ligation are designed, and comprise a first DNA fragment and / or a second DNA fragment. According to the present invention, the designed DNA fragments cannot be self-ligated, and have relatively good directivity on ligation with the to-be-detected DNA; after subjected to enzyme digestion, the to-be-detected DNA is ligated with the fragments for DNA ligation under the effect of the DNA ligase. and thus, a structure of the first DNA fragment-to-be-detected DNA-second DNA fragment is formed according to a sequence from a 3' end to a 5' end. The technology of the invention is especially suitable for ligation of trace DNA, DNA quantitative determination and construction of a sequencing library.

Description

technical field [0001] The present invention relates to a DNA connection method and its application, in particular to a method especially suitable for trace DNA connection, DNA quantitative determination and sequencing library construction, belonging to the field of DNA detection. Background technique [0002] In the research of nucleic acid, it is often necessary to link two or more nucleic acid fragments, for example, to connect a linker on a DNA molecule. The linker is generally a short blunt-ended or sticky-ended artificially synthesized nucleotide fragment. The ligation efficiency of adapters and nucleotides directly affects the quality of sequencing library construction, and currently there is no feasible method for ligation of trace DNA. [0003] Accurate quantification of DNA copy number is one of the important applications of modern molecular biology and medicine. Common DNA quantification methods mainly include UV spectrophotometry, fluorescent dye method, polymer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B50/06C12Q1/6851
CPCC12N15/11C40B50/06C12Q1/6851C12Q2521/301C12Q2521/501C12Q2531/113Y02A50/30
Inventor 杨神州万成任军
Owner XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD