Unlock instant, AI-driven research and patent intelligence for your innovation.
A method for in vitro seamless assembly of macromolecular DNA
What is Al technical title?
Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A macromolecular, seamless technology, applied in DNA preparation, recombinant DNA technology, fermentation, etc., to achieve high sequence accuracy, controllable assembly success rate, and reduce difficulty
Active Publication Date: 2020-07-28
HUNAN HYBRID RICE RES CENT +1
View PDF3 Cites 0 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
Problems solved by technology
At present, there is no related method reported to achieve the goal of seamless assembly of macromolecular DNA through the "segmentation + step-by-step enzymatic assembly" mode
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
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
Examples
Experimental program
Comparison scheme
Effect test
Embodiment 1
[0029] The DNA sequence (seq1) of Nipponbare's Chr.3:3700958-3705941 was assembled from the 5'→3' direction by cutting the plasmid with a restriction endonuclease that produces cohesive ends, and the sequence length is 4.98kb. Proceed as follows:
[0030] 1. Using DNAssit 2.0 software to analyze the restriction sites of the 4.98kb DNA sequence, there are a total of 44 restriction sites that do not appear in the DNA sequence, including 4 common restriction sites, namely BamHI and PstI , SpeI and XbaI ( figure 1 ).
[0031] 2. Select the BamHI enzyme-cut backbone vector pLDR ( figure 2 ), then GGATC or GATCC can be selected for the splicing base of the assembled sequence, but since GGATC is relatively evenly distributed in the 4.98kb DNA sequence, GGATC is selected as the splicing base, and the corresponding assembly direction is 5′→3 'direction.
[0032] 3. There are 6 GGATC splicing bases in the 4.98kb DNA sequence, and two of them are selected to divide the 4.98kb DNA se...
Embodiment 2
[0041] The DNA sequence (seq2) of Nipponbare's Chr.9:16782917-16790009 was assembled from the 3'→5' direction by cutting the plasmid with a blunt-ended restriction endonuclease, and the sequence length was 7.09kb. Proceed as follows:
[0042] 1. Using DNAssit 2.0 software to analyze the restriction sites of the 4.98kb DNA sequence, a total of 26 restriction sites do not appear in the DNA sequence, including 8 blunt-ended restriction sites, namely FspAI, PmeI, SmaI, SnaBI, SrfI, SspI, SwaI and XmnI ( Figure 7 ).
[0043] 2. Select the commonly used SmaI enzyme-cut backbone vector pLDR, then the splicing base of the assembled sequence can be CCC or GGG. Since the assembly direction is 3'→5' direction, GGG is selected as the splicing base.
[0044] 3. There are multiple GGG splicing bases in the 7.09kb DNA sequence. Select one of the splicing bases in the middle of the sequence to divide the 7.09kb sequence into two DNA fragments PF1 and PF2 of similar size. The lengths are 3...
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
Login to View More
Abstract
The invention provides a method for in vitro seamless assembly of large molecular weight DNAs. The method capable of achieving the seamless assembly of the large molecular weight DNAs through a 'subsection + stepped enzymatic assembly' mode has the advantages that 1. Splicing base groups are utilized to divide large-segment DNA molecules into a plurality of small segments, PCR difficulty is reduced, and the goal of substep seamless assembly is achieved due to existence of the splicing base groups. 2. Compared with a conventional enzyme-cut and link up method, the method does not rely on linkage and is hardly subjected to limitation of digestion locus. 3. A shortcoming that an extra base group is introduced easily in the conventional enzyme-cut and link up method and a locus specificity recombination method, and the goal of substep seamless connection is achieved. 4. The method has more controllable assembly success rate and higher sequence accuracy rate than one-step clone enzymatic assembly of multiple segments.
Description
technical field [0001] The invention relates to the technical field of DNA recombination in vitro, in particular to a method for seamless step-by-step assembly and connection of macromolecular DNA. Background technique [0002] Synthesis of macromolecular DNA fragments by DNA recombination technology in vitro has more and more demands in the study of gene function. One of the most basic requirements for the synthesis of large fragments is to assemble multiple small DNA fragments in a certain direction and precise base sequence to form larger DNA molecules. Traditional methods rely on restriction enzyme digestion and DNA ligase ligation. In vector construction, this method is generally suitable for the insertion of DNA fragments less than 3kb. Once the inserted DNA molecule is too large, there will often be no suitable enzyme cutting site for connecting the insert fragment and the vector. Sites for segmental digestion and ligation will introduce unnecessary restriction site...
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
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to View More 
Login to View More