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One-tube reaction type DNA molecule clone splicing method

A technology of DNA molecules and reactions, applied in the field of molecular biology, can solve the problems of low efficiency, failure to realize the standardization and reuse of DNA fragments, and the existence of DNA tail sequence modification, so as to avoid costs and improve the efficiency of ligation reactions

Active Publication Date: 2017-04-26
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the standardization and reuse of DNA fragments are still not realized, and the sequence modification at the end of DNA still exists
In addition, the multi-fragment DNA connection is still not reliable enough, the efficiency of assembly of more than four fragments is low, and the number of transformants is very rare

Method used

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  • One-tube reaction type DNA molecule clone splicing method
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  • One-tube reaction type DNA molecule clone splicing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The optimization of embodiment 1 thermocycling program

[0053] The six different thermal cycle programs described in Table 3 are used for the method of gene splicing described in the present invention (except that the thermal cycle program is different, all the other experimental materials and methods are the same), the results show that: longer reaction time, more The number of reaction cycles, lower annealing temperature and reaction temperature are beneficial to the experiment. There was no significant difference in the results of two-step thermocycling adjustments and three-step cycling conditions (Table 3, figure 1 ). Therefore, in conjunction with the PCR reaction, three-step thermal cycle conditions, longer reaction time, and more reaction cycles are used to improve reaction efficiency.

[0054] table 3

[0055] group Reaction conditions; number of cycles number of transformants 1 55C 15s, 65C 45s; 20 times 36 2 55C 15s, 65C 45s; 30...

Embodiment 2

[0061] The optimization of embodiment 2 bridging primers

[0062] 1. Design optimization of bridging primers

[0063]The bridging primer of the present invention is composed of two half bridging primers (bridge primer 1 and bridging primer 2), wherein bridging primer 1 is complementary to the 5' phosphorylated end of a target DNA; bridging primer 2 is complementary to the 3' phosphorylated end of another target DNA The phosphorylated ends are complementary, and the bridging primer 1 and bridging primer 2 form a continuous bridging primer. The present invention sets up two groups for the design of bridging primers, and the first group is to design bridging primer 1 and bridging primer with the same Tm value 2; The second group is to design bridging primer 1 and bridging primer 2 with the same length, and use these two groups to carry out the method for gene splicing of the present invention (except that the bridging primer design is different, the rest of the experimental mater...

Embodiment 3

[0068] The optimization of embodiment 3 reaction buffer components

[0069] The thermal cycle conditions used in the experiment are the conditions shown in Table 4, and the reaction system is shown in Table 5.

[0070] Table 5 reaction system

[0071]

[0072] 1. Since one tube reaction contains two enzymes, Taq ligase and T4 polynucleotide kinase, different enzymes will use different buffer components. This experiment takes into account the combination of two different enzyme components The formed buffer components (20X) were added to the final reaction system; other experimental groups were Taq ligase buffer, T4 polynucleotide kinase buffer or pure water.

[0073] After testing, it was found that the effect caused by different buffers is very obvious. Except for the experimental group in which the two buffers were simply mixed, the buffers used in the other experimental groups resulted in the final number of transformants being 0 (such as Figure 5 , during the experim...

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Abstract

The invention provides a one-tube reaction type DNA molecule clone splicing or reconstructing method. A reaction system is formed by mixing a target DNA fragment, a bridging primer, polynucleotide kinase, a heat stability ligase and a reaction buffer solution, the buffer solution contains ATP, NAD<+> and Mg<2+>, the pH value is 6-10, and the method is completed through a heat cycle process. The method realizes one-shot completion of a linking reaction in one tube, greatly improves the linking connection efficiency, and also has the following advantages: 1, phosphorylation and splicing steps of the DNA fragment are completed in one tube through one step to realize the linking reaction, so high cost caused by use of a phosphorylation primer is avoided; 2, the DNA fragment is seamlessly linked, so no superfluous nucleotides are introduced; 3, DNA sequence dependence is avoided; 4, not other nucleotides are introduced to the tail end of the spliced DNA fragment, and the DNA fragment can be reused for other construction works; and 5, the price is low, and is lower than that of routine enzyme digestion linking methods.

Description

technical field [0001] The invention relates to the technical field of molecular biology, more specifically, to a one-tube reaction type DNA molecular cloning splicing method. Background technique [0002] DNA splicing technology is one of the most important technical methods in current biological research, and most of molecular biology and cell biology are based on this technology. However, since Cohen and other researchers recombined the first DNA molecule 40 years ago, the mainstream DNA manipulation method is still based on the original enzyme-cut ligation method. There are 6 bp nucleotide scars left in the generated DNA products; at the same time, this traditional method cannot meet the complex and diverse application requirements of today. For example, multi-fragment ligation in metabolic engineering, standardization and reuse of DNA fragments in synthetic biology, and efficient and reliable splicing of DNA fragments of different sizes. [0003] Based on the above ge...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12P19/34
CPCC12N15/1027C12P19/34
Inventor 陆勇军阿迪亚
Owner SUN YAT SEN UNIV
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