Unlock instant, AI-driven research and patent intelligence for your innovation.

A vector insertion position detection method based on second-generation genome sequencing

A technology of whole genome sequencing and insertion position, which is applied in genomics, microbial measurement/inspection, biochemical equipment and methods, etc. It can solve problems such as false positives, poor detection, time-consuming and labor-intensive, etc., to achieve accurate results, The effect of fast processing speed and shortened analysis time

Inactive Publication Date: 2019-01-15
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The product of TAIL-PCR can be directly used for sequencing, but in many cases, the test is not very good. A better method is to perform TA cloning of the PCR product and then perform sequencing. There are false positives in the clone
TAIL-PCR, and construction of clone sequencing, time-consuming and laborious

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
  • A vector insertion position detection method based on second-generation genome sequencing
  • A vector insertion position detection method based on second-generation genome sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the acquisition of vector insertion mutation material

[0030] 1. Using rice kittake seeds as raw materials, transgene them with EAR4 / VP64 vectors, the method is as follows:

[0031] (1) Preparation of transformed receptors: select immature embryos 14 days after pollination, or embryogenic callus (1 mm) naturally dispersed from immature embryos and bright yellow in color as receptors for Agrobacterium infection.

[0032] (2) Cultivation of Agrobacterium engineering strains: Get a small amount of bacterial liquid from the Agrobacterium stock solution (20% glycerol), mark it on the YEB solid medium (containing kanamycin 50mg / l and rifamycin 50mg / l) Line culture, culture at 28°C in the dark until a single colony with a diameter of 1 mm grows. Take a single colony and inoculate it on the same medium, and cultivate it until the vigorous growth period. Pick a little bacterium and inoculate in 20ml YEB liquid medium (containing corresponding antibiotics), shake...

Embodiment 2

[0041] Embodiment 2, detection and analysis of vector insertion position

[0042] 1. The whole genome of the mutant Mu2 obtained in Example 1 was sequenced using next-generation sequencing technology. Next-generation sequencing technologies such as Roch's 454 technology, illumina's Solexa, Hiseq technology, and ABI's Solid technology can be used for whole-genome sequencing.

[0043] 2. After completing step 1, use the bowtie2 comparison software for comparison, the method is as follows:

[0044] (1) All the complete reads sequenced in step 1 were excised from the 5'-3' direction to 2 / 3 of the length of the sequence, and the remaining partial sequences were aligned to the EAR4 / VP64 vector, and the ones that could be 100% aligned to the vector were screened All reads sequences;

[0045] (2) Align all the complete reads obtained in step (1) from the 2 / 3 length sequence in the 3'-5' direction, and align the remaining partial sequences to the rice Nipponbare reference genome, and...

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 vector insertion position detection method based on genome second generation sequencing. The method comprises the steps of sequencing the whole genome of the plant mutant andperforming sequence analysis by using sequence alignment software according to a specific method to obtain an insertion position of a vector. The method has the following advantages: (1) a server isused for analysis, the processing speed is fast, and each sample can be completed in about half an hour, compared with at least 3 days of TAIL-PCR methods, the analysis time is greatly shortened. (2)the results are accurate and specific by using high-quality reads, but TAIL-The principle of PCR method is to amplify PCR products by annexing nonspecific primers, and there are some false positives.

Description

technical field [0001] The invention relates to a method for detecting the insertion position of a vector based on second-generation sequencing of the genome. Background technique [0002] Vector insertion mutagenesis plays an important role in the study of plant functional genomics and is widely used in the analysis of plant genome functions. It is an effective tool for analyzing new genes and studying gene functions. Now there are T-DNA insertion libraries and overexpression vector libraries on the market. Knowing the position of vector insertion is the first problem to be solved by the vector insertion library, detecting whether the vector is inserted into the gene to cause mutation, etc. Among them, TAIL-PCR is the mainstream method for detecting the position of vector insertion. Symmetric PCR is a chromosome walking technique. This technology uses three nested specific primers and degenerate primers to carry out continuous PCR cycles, using different annealing tempera...

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): C12Q1/6858G16B20/50
CPCC12Q1/6858C12Q2535/122C12Q2537/165
Inventor 袁运栋王永红
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI