Method for constructing a DNA library and application thereof

A technology for DNA library and end repair, which is applied in the kit for constructing DNA library and in the field of constructing DNA library, which can solve the problems of long time for library construction, low conversion efficiency, and method needs to be improved, and achieve high efficiency of end repair and tailing , low preference, shortened reaction time and the effect of reaction flow

Active Publication Date: 2020-04-28
ZHEJIANG ANNOROAD BIO TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The library construction takes a long time, usually more than two hours, and the transformation efficiency is low
[0004] Therefore, the method of constructing DNA library needs to be improved

Method used

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  • Method for constructing a DNA library and application thereof
  • Method for constructing a DNA library and application thereof
  • Method for constructing a DNA library and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] In this embodiment, the reaction conditions for end repair and end protrusion adenylation are optimized, as follows:

[0065] Since adenylation enzymes usually show a certain preference, DNA fragments with pyrimidine (dT or dC) at the 3'end are more likely to be adenylated than DNA fragments with purines (dA or dG) at the 3'end. Incomplete adenylation will lead to The blunt-end ligation reaction between DNA fragments affects the genome sequence assembly of the sequencing data. In order to improve this, on the basis of the polymerase basic buffer system, the pH was increased to 8.8, and the magnesium ion concentration was adjusted to 10mM to form a modified buffer suitable for one-step end repair and A-tailing (10mM Tris-HCl, 10mM MgCl2, 50mM KCl, 0.1% Triton-x-100, PH8.8), as shown in Table 1 and figure 1 As shown, compared with the existing blue buffer (Blue buffer), the reaction efficiency of terminal adenylation is improved and preference is reduced.

[0066] In this exa...

Embodiment 2

[0072] Using the modified buffer of Example 1, the influence of the temperature and time conditions of the adenylate tailing reaction on the yield of the library was explored, the amount of enzyme, and the reaction time and temperature were as follows: image 3 with 4 As shown, specifically, the concentrations of the modified Taq DNA polymerase are 0.1U, 0.2U, 0.4U, and 1U, respectively, and the reaction time and temperature of the adenylate tail reaction are 68 degrees Celsius, 10 minutes; 68 degrees Celsius, 30 minutes ; 72 degrees Celsius, 10 minutes; 72 degrees Celsius, 30 minutes; 75 degrees Celsius, 10 minutes; 75 degrees Celsius, 30 minutes. At the same time, 100ng digested DNA fragments were used as substrates to explore the usage of modified Taq DNA polymerase.

[0073] The result is image 3 with 4 Shown in image 3 In, the modified buffer and modified Taq DNA polymerase of Example 1 were used in one-step end repair and adenylate addition reaction. The amount of substrat...

Embodiment 3

[0075] In the modified buffer of Example 1, add interruption buffer (0.1×DnaseI Buffer, 1×BlueBuffer, 50mM Nacl, 0.1% TritonX-100, 120ng sso7d), and use NA12878 standard substrate to build in this buffer system. The library is evaluated for the compatibility of DNA fragmentation enzymes. The amount of substrate and library yield are shown in Table 2.

[0076] Table 2

[0077]

[0078] The conditions for interrupting the integration of library construction are 37°C for 10 minutes and 72°C for 20 minutes. Reactions 2 and 3 are repetitions of reaction 1, and the results are as follows Figure 5 Shown.

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Abstract

The invention discloses a method for constructing a DNA library and application thereof. The method comprises the following steps: carrying out terminal repairing on a DNA sample, and carrying out adenine nucleotide tailing treatment on the DNA sample by using high-temperature-resistant polymerase so as to obtain a tailed DNA under the condition of performing the terminal repairing and the adeninenucleotide tailing treatment continuously; and performing linker ligation treatment on the tailed DNA so as to obtain a linking product. In the DNA library, the conditions of terminal repairing and adenine nucleotide tailing treatment are as follows: the temperature is 27-37 DEG C and the time is 5-30 minutes; and the temperature is 70-75 DEG C, and the time is 10-20 minutes. According to the method, tail end repairing and adenine nucleotide tail adding treatment are continuously carried out, purification treatment is not needed in the process, stability is good; meanwhile, reaction time is remarkably shortened, and the reaction process is remarkably simplified.

Description

Technical field [0001] The present invention relates to the field of gene sequencing, in particular to a method for constructing a DNA library and a kit for constructing a DNA library. Background technique [0002] Second-generation sequencing and corresponding bioinformatics analysis of genomic DNA can realize chromosomal abnormality diagnosis, genetic detection of related disease genes, genome resequencing, exome sequencing and other related medical and health fields and scientific and technological service fields, and the scope of application widely. [0003] The general process of constructing a second-generation sequencing library is as follows: fragment the target DNA; blunt the free DNA; protrude adenylation at the 3'end of the blunt DNA; protrude the adenylated DNA The fragment is connected to a double-stranded Y-linker that highlights thymidine. The library construction time is long, usually more than two hours, and the conversion efficiency is low. [0004] Therefore, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C40B50/06
CPCC12Q1/6806C40B50/06C12Q2525/191C12Q2521/501
Inventor 潘伟业王亚蕾程世月李大为玄兆玲王海良王娟肖飞
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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