Check patentability & draft patents in minutes with Patsnap Eureka AI!

Tobacco BAC library and construction and quality detection methods thereof

A quality detection method, the technology of tobacco, applied in the field of molecular biology, can solve the problems of difficult complete separation of large fragments of DNA, affecting the quality of BAC library, false positives, etc.

Pending Publication Date: 2019-11-22
YUNNAN ACAD OF TOBACCO AGRI SCI
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because early library construction was limited by technology and cost, high-quality, large-capacity BAC library construction work was rarely carried out, so the specific parameters, detection standards, and quality evaluation of these BAC libraries were rarely reported
[0003] In the existing BAC library construction technology, an appropriate concentration of genomic DNA is a necessary condition for obtaining high-quality large fragments of DNA. It is often difficult to successfully obtain large fragments of DNA to construct a BAC library at a low concentration of genomic DNA, and the concentration of genomic DNA is too high. It will increase the difficulty of the experiment. For example, it will be difficult to completely separate large fragments of DNA in the pulsed field. Many small fragments of DNA and large fragments of DNA cannot be separated by PFGE, and DNA accumulates together.
Because the larger the exogenous DNA fragment is, the more difficult it is to connect to the BAC carrier, but under the same conditions, small fragments of exogenous DNA are easily connected to the carrier, and there will be a lot of small fragments in the constructed BAC library, which will seriously affect the BAC library. the quality of
Insert fragments with high molecular weight and uniform size are also one of the key links in the construction of BAC libraries. If the endonuclease is excessive in the experiment, it will lead to non-specific digestion and a high proportion of false positives, resulting in the empty load rate of the entire library. too high

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Tobacco BAC library and construction and quality detection methods thereof
  • Tobacco BAC library and construction and quality detection methods thereof
  • Tobacco BAC library and construction and quality detection methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Extracting large fragment DNA from tobacco.

[0053] Using Wz with high resistance to tobacco black shank as the donor parent, combined with marker selection, this resistance was introduced into the main flue-cured tobacco variety K326 in my country through backcrossing method, and BC7F7 was obtained. Among them, 14-60 with excellent agronomic traits was selected as Materials for this library construction.

[0054] Tobacco seeds grow to the 4-leaf stage, collect young leaves, add liquid nitrogen to grind, separate the nucleus, and then mix with an equal volume of 1% low-melting point agarose to make nucleus embedding blocks, treat with proteinase K, and store in 4 ℃ in TE for later use.

Embodiment 2

[0055] Example 2: DNA fragment screening.

[0056] The first screening: the obtained tobacco DNA embedding blocks were subjected to 16 enzyme digestion reactions. Enzyme digestion reaction system: 25 μL H 2 O, 10 μL 10×buffer, 0.4 nmol spermidine, 5 μL 1.0 U / μL Hin dIII enzyme and chopped 1 / 2 tobacco DNA embedding block, put the reaction system in a constant temperature water bath at 37 ℃, and react for 15 min. Large fragments of DNA were then separated by pulse electrophoresis.

[0057] like figure 1 Shown: After electrophoresis, cut the gel block into 3 parts at 0.5 cm inwards on both sides of the sample hole, stain the gel blocks on both sides with ethidium bromide solution, rinse and put it into the gel imager Perform imaging analysis using a transparent ruler as a reference. According to the results of gel imaging, cut out a strip of 100-250 kb with a width of about 2 cm from the middle of the unstained gel, and divide the strip into two small gels with a width of a...

Embodiment 3

[0058] Example 3: Preparation of pIndigoBAC536-S vector.

[0059] use Hin Digest the pHZAUBAC1 vector plasmid with dIII restriction endonuclease. The digestion reaction system is: 2 μL of pHZAUBAC1 carrier DNA, 15 μL of 10×buffer R for HindIII, 2 μL of HindIII restriction enzyme, and 1 μL of ddH2O 13 .

[0060] After the digestion reaction, two fragments, pIndigoBAC536-S and pGEM-4Z, were obtained. Add CIAP to the product to dephosphorylate it, then recover it by chloroform / isoamyl alcohol extraction, then separate the two carrier fragments by pulse-field gel electrophoresis, and finally obtain the pIndigoBAC536-S vector by gel cutting recovery and electroelution.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a tobacco BAC library and construction and quality detection methods thereof. According to the tobacco BAC library, a near-isogenic line 14-60 of a tobacco variety K236 containing a high black shank resistance gene is used as a material for extracting genome DNA; pIndigoBAC536-S is used as a vector, Escherichia coli DH10B is used as a host, the library contains 414720 monoclones, the size of an inserted DNA fragment is 97-160 kb, and the library covers a tobacco genome by 11 times or above. The tobacco BAC library disclosed by the invention can well cover genome information of tobacco, can be used as an important resource for improving and perfecting a tobacco genome sequence, and also provides an important resource for further cloning important functional genes ofthe tobacco and researching a new tobacco antibody source.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a tobacco BAC library and its construction and quality detection methods. Background technique [0002] The BAC library is a genomic library with an insert fragment of 50-300 kb constructed on the basis of a bacterial artificial chromosome vector, and each clone contains all the gene information of a part of the chromosome. The BAC library is an important tool for genome structure research and functional gene isolation, and has played an important role in physical map construction, functional gene map cloning, genome full sequence determination, and comparative genomics analysis. BAC libraries have been widely used in the research of various species, including bacteria, animals and various crops. There are few reports on the construction of tobacco BAC library, only the wild species of Nicotiana tomentosa, Nicotiana Hicks Broadleaf and SR1. Because early l...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C40B50/06C12N15/66C12Q1/6869
CPCC40B40/06C40B50/06C12N15/66C12Q1/6869
Inventor 曾建敏刘勇黄昌军袁诚于海芹方敦煌宋中邦李永平董庆园马德清
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com