WO2015078272A1

WO2015078272A1 - High-throughput vector construction method

Info

Publication number: WO2015078272A1
Application number: PCT/CN2014/090368
Authority: WO
Inventors: 张玮; 蒙伟能; 温华杰; 李春园; 王文忠; 施金秀; 魏宝丽
Original assignee: 赛业（广州）生物科技有限公司
Priority date: 2013-11-26
Filing date: 2014-11-05
Publication date: 2015-06-04
Also published as: CN103602697A

Abstract

Provided in the present invention is a method for constructing a high-throughput vector based on Gateway technology, wherein the method comprises the steps of a high-throughput PCR amplification, BP reaction, BP cloning, monoclonal culture, plasmid extraction, LR reaction and LR cloning etc. The BP reaction uses a pDoner221 plasmid, and the LR reaction uses three plasmids, wherein the first plasmid may be a p4P1R plasmid, the second plasmid may be a P2RP3 plasmid, and the third plasmid may be a PDest plasmid.

Description

High-throughput carrier construction method

Technical field

The present invention relates to a high throughput vector construction method.

Background technique

With the rapid development of biotechnology, the continuous breakthroughs in the field of related biological research and the emergence of new research fields, carrier construction work is becoming more and more common. Vector construction involves DNA nucleic acid sequence analysis, protein function analysis, genetic genetic analysis, and the like, including: genetics, bioinformatics, proteomics, protein functioning and other fields of almost all biological related research and production. At present, the construction of vectors is mainly based on enzymatic cleavage, and single-sample single-channel operation is the main. When constructing a multi-component vector, it is often difficult to construct a vector because a suitable restriction site cannot be found. Often because of the single-channel vector construction, the vector construction efficiency and scale cannot be improved. Therefore, a variety of nucleic acid manipulation methods are needed to achieve the purpose of vector construction. This results in a large consumption of financial, human and material resources. At the same time, the construction of single-channel carriers often requires a lot of repetitive work. The duplication and disorder of work often lead to cross-contamination of samples in the construction of the carrier, which causes great inconvenience to the work. In addition, with the rapid development of bioinformatics, the current high-throughput research work is gradually increasing. In the face of high-throughput carrier construction, high-throughput work often delays time due to small flux.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the above deficiencies and provide a high-throughput carrier construction process based on Gateway technology. To achieve the above objects, the present invention provides a high throughput vector construction method.

The technical solution adopted by the invention to solve the technical problem is: a high-throughput carrier structure A method is constructed, the method comprising the steps of:

(1) adding the target gene to be captured of different templates to a 96-well PCR plate for high-throughput PCR for purification;

(2) determining the concentration of the purified PCR product;

(3) transferring the product to a 96-well BP reaction precast plate, and performing BP reaction with the BP reaction mixture disposed in the 96-well BP reaction precast plate;

(4) transferring the BP product into a competent cell plate for cloning transformation of the BP product;

(5) transferring the transformed BP cells to a large LB solid medium, and drawing a line to separate the monoclonal;

(6) The BP reaction was picked up by monoclonal, and then transferred to a 96-well PCR clone to identify the pre-formed plate for cloning and identification of the BP product;

(7) The identified positive clones were transferred to a 96-deep well plate. After culture, the BP clone plasmid was extracted by 96-well plasmid extraction, and the BP clone plasmid concentration was determined.

(8) Transfer the product to a 96-well LR reaction precast plate for LR reaction;

(9) transferring the LR product into a competent cell plate for LR product transformation;

(10) transferring the transformed LR product to a large LB solid medium for line drawing separation of the monoclonal;

(11) The LR reaction monoclonal was picked and cultured, and then transferred to a 96-well PCR clone to identify a pre-made plate for cloning and identification of the LR product;

(12) The identified LR positive clones were transferred to 96 deep well plates, and the LR product cloning plasmid was extracted after the culture, and the LR cloning plasmid concentration was determined.

Preferably, said step (2) is carried out using microbeads.

Preferably, the microbeads are magnetic microbeads.

Preferably, each portion of the 96-well BP reaction pre-formed BP reaction mixture comprises the following weight components:

BP enzyme 1 μL (1 U/μL),

pDoner221 plasmid 1 μL (75 ng/μL),

Buffer eluate 2 μL;

Each of the BP reaction mixtures was reacted with 1 μL (20 ng/μL) of the purified PCR product.

Preferably, each portion of the LR reaction mixture of the 96-well LR reaction preform is comprised of the following weight components:

Preferably, the first plasmid is a P4P1R plasmid, the second plasmid is a P2RP3 plasmid, and the third plasmid is Pdest.

Preferably, in the steps (4) and (9), the competent cell plate is a 96-well competent cell plate.

Preferably, in the steps (5) and (10), the separated monoclonals are separated by a line drawing method.

Preferably, in steps (7) and (12), the cloning plasmid is extracted by a 96-well plate plasmid extraction method.

The invention has the beneficial effects that the Gateway vector construction method and related products involved are applied to high-throughput carrier construction, and the method achieves high throughput, intensification, high efficiency and low compared with the current single carrier construction. Cost and other advantages.

DRAWINGS

Further description of the invention will be made in the following with reference to the accompanying drawings and embodiments.

Figure 1 is a schematic flow diagram of the creation of the present invention.

detailed description

The invention will now be described in further detail with reference to the accompanying drawings. The drawings are simplified schematic diagrams, only to illustrate the basic structure created by the present invention in a schematic manner, and thus only show the configuration related to the creation of the present invention.

A high-throughput vector construction method as shown in Figure 1 includes the following steps: 96-well PCR amplification of the target gene, 96-well PCR product purification, 96-well PCR product concentration determination, 96-well BP reaction, 96-well BP clone, Cloning and PCR monoclonal identification, 96-well BP monoclonal culture, 96-well BP monoclonal plasmid extraction, 96-well BP monoclonal plasmid concentration determination, 96-well LR reaction, 96-well LR cloning, cloning and PCR monoclonal identification, 96-well LR monoclonal culture, 96-well LR monoclonal plasmid extraction, 96-well LR monoclonal plasmid concentration determination.

Specific reaction steps include:

(1) The target genes to be captured of different templates were separately added to a 96-well PCR plate, and high-throughput PCR was performed to purify the PCR product.

Specifically, the PCR product can be purified by adding a magnetic microbead to a 96-well PCR plate and a 96-well magnetic frame method to homogenize the PCR product by unifying the Tm value and extension time of different template primers.

(2) The concentration of the product after purification was measured.

Specifically, the purified PCR product concentration on the 96-well microtiter plate was determined by high throughput using the Pico green method.

(3) The product was transferred to a 96-well BP reaction precast plate, and BP reaction was carried out with a BP reaction mixture disposed in the 96-well BP reaction precast plate.

Specifically, the BP reaction mixture of each of the 96-well BP reaction preforms comprises the following weight components:

BP enzyme 1 μL (1 U/μL),

pDoner221 plasmid 1 μL (75 ng/μL),

Buffer eluate 2 μL;

The total amount of each component of the BP reaction was 5 μL.

Specifically, the BP enzyme composition consists of two enzymes, IHF (molecular weight: α chain 11224.79 Da, β chain 10650.14 Da) and Int (molecular weight: 67090.86 Da). Preferably, the BP enzyme can be a BP enzyme produced by Life Technologies (name: Gateway BP Clonase II Enzyme mix, Cat. No. 11789-100). More preferably, the BP enzyme is a BP reaction recombinase produced by Saiye (Guangzhou) Biotechnology Co., Ltd.

The pDoner221 plasmid was obtained from MultiSite Gateway ThreeFragment Vector Construction Kit (Life technologies. Cat. No. 12537-023.)

The buffer eluate consists of: Tris-HCl 10 mM,

EDTA PH=8.0 1mM;

(4) The BP product was transferred to a competent cell plate for BP product transformation.

(5) The cells transformed with the BP product were transferred to a large LB solid medium, and a single line was isolated for drawing.

Specifically, high-throughput cloning, isolation and identification were carried out by a 96-well cloning method, a multi-channel line drawing method, and a 96-well PCR cloning method.

The BP product clone was transformed into 96 BP reaction products in one time using 96-well competent prefabricated plates. High-throughput segregation of monoclonals using large LB culture dishes and scribing methods, high-throughput identification of monoclonals using pre-made 96-well PCR mixtures. Positive clones were amplified by 96-well plate culture.

(6) The BP reaction was picked and cloned, and then transferred to a 96-well PCR clone to identify the pre-formed plate for cloning and identification of the BP product.

(7) The identified positive clones were transferred to a 96-deep well plate, and the BP clone plasmid was extracted after the culture, and the BP clone plasmid concentration was determined.

Specifically, the high-throughput plasmid extraction method was used to carry out the extraction of BP cloning plasmids with high throughput, and the concentration of BP cloning plasmid was determined by high-throughput method by Pico Green detection method. The concentration was determined by fluorescence microplate reader and Pico Green, and the plasmid concentration was determined by high throughput. High-throughput trim was 11.72 ng/μL per sample.

(8) The product was transferred to a 96-well LR reaction precast plate for LR reaction.

Specifically, a high throughput LR reaction was performed by using a 96-well plate. A 96-well LR reaction plate containing reagents other than the BP cloning plasmid product.

Specifically, the LR reaction mixture of each of the 96-well LR reaction preforms comprises the following weight components:

Specifically, the LR enzyme composition consists of three enzymes: IHF (molecular weight: α chain 11224.79 Da, β chain 10650.14 Da) and Int (molecular weight: 67090.86 Da) and Xis (molecular weight: 40249.05 Da). The LR enzyme can be produced by Life Technologies LR enzyme (name: LRClonase II Plus enzyme, Cat. No. 12538-200). More preferably, the LR enzyme is an LR reaction recombinase produced by Saiye (Guangzhou) Biotechnology Co., Ltd.

The pDoner221 product plasmid was a pDoner221 plasmid recombinant with the gene of interest.

The P4P1R plasmid, the P2RP3 plasmid and the Pdest plasmid were obtained from the MultiSite Gateway ThreeFragment Vector Construction Kit (Life technologies. Cat. No. 12537-023.)

(9) The LR product was transferred to a 96-well competent cell plate for LR product transformation.

(10) The cells transformed with the LR product were transferred to a large LB solid medium for line-separating monoclonal.

(11) The LR reaction was picked up by monoclonal, and then transferred to a 96-well PCR clone to identify a pre-formed plate for cloning and identification of the LR product.

Specifically, high-throughput cloning, isolation and identification were carried out by a 96-well cloning method, a multi-channel line drawing method, and a 96-well PCR cloning method. High-throughput 96-well plates were used to clone high-throughput LR products.

Specifically, high-throughput plasmid extraction and high-throughput LR cloning plasmid extraction were performed. High-throughput assays for LR cloning plasmid concentration determination by the Pico Green method.

Example

96-well PCR: Design different primers with reference to the template DNA sequence of interest. All primers had a Tm value of 60 °C. The attB1 and attB2 site sequences were added to the 5' end of the positive and negative primers, respectively. In the PCR procedure, the annealing temperature was uniformly 60 °C. The extension time is based on the longest PCR band. 96 samples were separately added to a 96-well PCR template. Run the PCR program in a unified manner.

96-well PCR product purification: The PCR product was purified by magnetic bead purification.

96-well BP reaction: The BP reaction mixture was uniformly configured in a 96-well PCR plate. The solution configurations are shown in Table 1.

Table 1. List of various components of BP reaction

Transformation of 96-well BP reaction product: Competent cells were pre-cast into 96-well PCR plates, and the BP product was added to competent cells by multi-channel loading using a 12-channel pipette. Achieve centralized conversion. Resuscitation of 96 deep-well plates was used to concentrate the recovery of competent cells after transformation.

Cloning, Isolation, and Identification of 96-well BP Products: Using a 12-channel pipette, the cells were pipetted to separate the monoclonal bacteria by pipetting the LB plates. And incubated at a suitable temperature overnight. After the culture, the monoclonals were grown along the line traces, and 10 μL of the pipette tip was used, and the monoclonal was collected, and then placed in a 96-well PCR plate, 10 μL of liquid LB medium containing Kanamycin per well. Incubate at 37 ° C, 250 rpm / min for 1.5 hours. After the culture is completed, 2 μL of the culture solution is aspirated for positive PCR detection. The PCR product was positive for agarose gel electrophoresis.

96-well monoclonal culture: 96 deep well plates pre-formed with Kanamycin resistant 1 ml of LB liquid medium. 5 μL of the positive clone LB medium was added to the above 96 deep well plate. Incubate at 37 ° C, 250 rpm / min for 24 hours.

Plasmid extraction of 96-well BP product: The culture solution of the deep-well plate was collected by centrifugation for 24 hours, and the BP product plasmid was mainly extracted by alkaline lysis and column purification.

Determination of 96-well BP product concentration: The concentration of the BP product plasmid was determined by Pico Green and fluorescence microplate reader detection methods.

96-well LR reaction, 96-well LR product transformation, clonal isolation and identification, 96-well LR product plasmid extraction, 96-well LR product plasmid concentration determination: The other four methods were the same as the BP reaction.

96-well LR reaction: The LR reaction mixture was uniformly configured in a 96-well PCR plate. The added LR enzyme is a patented LR enzyme developed by Saiye Bio. The solution configurations are shown in Table 2. Table 2. List of various components of LR reaction

In view of the above-described embodiments of the present invention, it is apparent that various changes and modifications can be made by those skilled in the art without departing from the scope of the invention. The technical scope of the invention is not limited to the contents of the specification, and the technical scope thereof must be determined according to the scope of the claims.

Claims

A high-throughput carrier construction method, characterized in that the method comprises the following steps:

(1) adding the target gene to be captured of different templates to a 96-well PCR plate for high-throughput PCR for purification;

(2) determining the concentration of the purified product;

(3) transferring the product to a 96-well BP reaction precast plate, and performing BP reaction with the BP reaction mixture disposed in the 96-well BP reaction precast plate;

(4) transferring the BP product to a competent cell plate for transformation of the BP product;

(5) transferring the transformed BP cells to a large LB solid medium for line drawing separation of the monoclonal;

(6) The BP reaction was picked up by monoclonal, and then transferred to a 96-well PCR clone to identify the pre-formed plate for cloning and identification of the BP product;

(7) Transfer the positive clone after identification to 96 deep well plates, extract BP cloning plasmid after culture, and determine the concentration of BP cloning plasmid;

(8) Transfer the product to a 96-well LR reaction precast plate for LR reaction;

(9) transferring the LR product into a competent cell plate for LR product transformation;

(10) transferring the transformed LR product to a large LB solid medium for line drawing separation of the monoclonal;

(11) The LR reaction monoclonal was picked and cultured, and then transferred to a 96-well PCR clone to identify a pre-made plate for cloning and identification of the LR product;

(12) The identified LR positive clones were transferred to 96 deep well plates, and the LR product cloning plasmid was extracted after the culture, and the LR cloning plasmid concentration was determined.
The method of claim 1 wherein said step (1) is carried out using microbeads.
The method of claim 2 wherein said microbeads are magnetic microbeads.
A method according to any one of claims 1 to 3, wherein each of said 96-well BP reaction pre-formed BP reaction mixture comprises the following weight components:

BP enzyme 1 μL (1 U/μL),

pDoner221 plasmid 1 μL (75 ng/μL),

Buffer eluate 2 μL;

Each of the BP reaction mixtures was reacted with 1 μL (20 ng/μL) of the purified PCR product.
The method of claim 4 wherein each of said 96-well LR reaction preformed LR reaction mixture comprises the following weight components:
The method according to claim 5, wherein the first plasmid is a P4P1R plasmid, the second plasmid is a P2RP3 plasmid, and the third plasmid is a PDest plasmid.
The method of claim 6 wherein said PDest plasmid is a viral backbone plasmid.
The method according to claim 6, wherein in the steps (4) and (9), the competent cell plate is a 96-well competent cell plate.
The method according to claim 7, wherein in the steps (5) and (10), the separated monoclonals are separated by a line drawing method.
The method according to claim 8, wherein in the steps (7) and (12), the cloning plasmid is extracted by a 96-well plate plasmid extraction method.