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

Method for efficiently and quickly determining BAC (bacterial artificial chromosome) terminal sequence

A technology of terminal sequence and sequencing platform, applied in the field of efficient and rapid determination of BAC terminal sequence, can solve the problems of low success rate, low throughput, complicated operation, etc., and achieve the effect of reducing cost and time

Active Publication Date: 2014-10-01
CHINA AGRI UNIV
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, BAC terminal sequencing is based on Sanger technology, which is similar to the sequencing steps of plasmids or PCR products, but the success rate is much lower than them, mainly because it is difficult to obtain a large amount of high-purity BAC DNA (above 500ng) and the operation is complicated , low flux, high cost

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
  • Method for efficiently and quickly determining BAC (bacterial artificial chromosome) terminal sequence
  • Method for efficiently and quickly determining BAC (bacterial artificial chromosome) terminal sequence
  • Method for efficiently and quickly determining BAC (bacterial artificial chromosome) terminal sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of a next-generation sequencing library containing all BAC terminal sequences

[0046] Select 16 secondary pools (including 3 plate pools, 5 row pools, and 8 column pools) of the No. 10 super pool DHS10 of the duck genome BAC library. The spatial positions of these 16 secondary pools are 3×5× 8 = 120 intersections. Use the Covaris S220 ultrasonic disruption system to break the BAC DNA into 400-500bp. After end repair, connect specific adapters at both ends, then amplify with specific PCR primers, purify and recover the amplified product of 300-350bp. That is to make a sequencing library containing all BAC terminal sequences of the secondary pool.

[0047] Specifically, the method for preparing the above-mentioned sequencing library containing all BAC end sequences of the secondary pool comprises the following steps:

[0048] (1) Break the BAC DNA

[0049] Add 5μg DNA to a 1.5ml centrifuge tube, dilute it with TE solution to make up its volume to...

Embodiment 2

[0066] Example 2 Using Ion Torrent PGM Sequencing

[0067] After library quality control and water-in-oil PCR reaction, sequencing was carried out on the machine according to the standard procedure of Ion Torrent PGM, and the original data file in the format of fastq was obtained.

Embodiment 3

[0068] Example 3 Returning mixed BAC end sequences to a single clone

[0069] By aligning the BAC end sequence obtained in the row pool with the BAC end sequence in the column pool and plate pool, the column pool and plate pool number where each BAC end sequence appears, and removing the repeated BAC end sequence, each BAC The clone to which the terminal sequence belongs, that is, the BAC terminal sequence of each clone is obtained. Theoretically, it should be possible to obtain the end sequences on both sides of 120 clones, that is, 240 BAC end sequences, and the actual analysis results also proved the prediction of the present invention.

[0070] At the same time, the results were verified using the BAC terminal sequencing method based on Sanger sequencing, and it was proved that the sequences obtained by the two were consistent, which proved the reliability of the present invention.

[0071] figure 1 The comparison result of the BAC terminal sequence (277bp) of a certain ...

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 provides a method for efficiently and quickly determining the BAC (bacterial artificial chromosome) terminal sequence. The method comprises the following steps: (1) with a super cell as a unit, enriching all BAC terminal sequences in the row cells, column cells and plate cells in each super cell to obtain a library suitable for a new-generation sequencing platform; (2) sequencing on the new-generation sequencing platform; and (3) regressing the mixed BAC terminal sequences to single cloning through the method of bioinformatics: determining the information of the row cell, column cell and plate cell of each BAC terminal sequence through sequence comparison, and positioning the BAC terminal sequence of each cloning. The method provided by the invention not only can quickly obtain the BAC terminal sequences of all single cloning in the whole BAC library, but also reduces the cost and time of BAC terminal sequencing to a great degree.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for quickly and efficiently determining the sequence of a BAC terminal. Background technique [0002] Genome BAC (Bacterial Artificial Chromosome, Bacterial Artificial Chromosome) library is a recombinant DNA clone population containing random fragments of the genome of an organism. It plays an important role in the construction of physical maps, gene map cloning, and gene structure analysis. Many organisms currently have high-coverage BAC libraries. In order to facilitate management, the BAC library is divided into multiple super pools for storage, and each super pool consists of a certain number of 96-well or 384-well plates; BAC clones are mixed to obtain row pools, column pools and plate pools. The 3D-PCR screening system is commonly used for screening clones. The first step is to perform PCR amplification on the DNA of the super pool to find out the super pool contain...

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
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2531/113C12Q2535/122
Inventor 胡晓湘谈成李宁
Owner CHINA AGRI UNIV