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Method for improving DNA cloning and assembling efficacies by utilizing short-chain primer repression

A short-chain, purpose-built technology that can be used in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., and can solve problems such as assembly efficiency that cannot be ignored

Active Publication Date: 2017-06-09
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the process of assembling long fragments of DNA, the efficiency of assembly cannot be ignored, that is, how many proportions of DNA fragments can be correctly connected into long fragments of interest

Method used

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  • Method for improving DNA cloning and assembling efficacies by utilizing short-chain primer repression
  • Method for improving DNA cloning and assembling efficacies by utilizing short-chain primer repression
  • Method for improving DNA cloning and assembling efficacies by utilizing short-chain primer repression

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Embodiment 1

[0125] Example 1. A method for improving DNA cloning and assembly efficiency by using short-chain primer suppression

[0126] The specific test process in this embodiment is as attached figure 2 shown.

[0127] 1. Preparation of 7 circular recombination vectors and receiving vector x

[0128] Circular recombination vector 1 includes DNA fragment A 1, DNA fragment 1 to be spliced, DNA fragment B 1 and backbone carrier from upstream (the nucleotide sequence of the backbone carrier is after removing nucleotides 4799 to 5663 in sequence 1 The obtained sequence); the DNA fragment A1 in the circular recombination vector 1 is the 23rd-55th position in the sequence 11, and the DNA fragment B1 is the 395th-426th position in the sequence 11;; the circular recombination vector 1 The nucleotide sequence of the DNA fragment A 1 that is identical or complementary to the short-chain primer A is the 30th-51st position in the sequence 11, the nucleotide sequence of the DNA fragment 1 to be ...

Embodiment 2

[0315] Example 2, the application of the method of short-chain primer suppression in the splicing of multiple fragments

[0316] The principle of this embodiment is attached Figure 4 shown.

[0317] 1. Preparation of 7 DNA fragments

[0318] The nucleotide sequence of DNA fragment 1 is shown as sequence 25 in the sequence table. DNA fragment 1 sequentially includes DNA fragment A1, DNA fragment 1 to be spliced ​​and DNA fragment B1; DNA fragment A1 and short chain in DNA fragment 1 The nucleotide sequence identical or complementary to primer C is the 27th-53rd nucleotide molecule in sequence 25; the nucleotide sequence of DNA fragment 1 to be spliced ​​is 54th-256th in sequence 25, DNA fragment B1 and The same or complementary nucleotide sequence of the short-chain primer D is the 257th-281th position in sequence 25;

[0319] The nucleotide sequence of DNA fragment 2 is shown as sequence 26 in the sequence table. DNA fragment 2 includes DNA fragment A 2, DNA fragment 2 to ...

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Abstract

The invention discloses a method for improving DNA cloning and assembling efficacies by utilizing short-chain primer repression. According to the method, two sections of DNA sequences are designed on the original carrier, so that the target DNA fragments are separated from a carrier framework sequence, restriction enzyme sites are designed at two sides of the two sections of DNA sequences, when DNA fragments are assembled, through enzyme digestion, the original carrier is divided into four parts, then a short-chain primer is added, the short-chain primer is subjected to complementary pairing with one chain in the DNA sequence added on the original carrier, after the repression process, the sticky ends of the target DNA fragments are not matched with the carrier, the target DNA fragments cut down can not be connected with the original carrier, so that the self-bonding process of the DNA fragments after enzyme digestion and the original carrier is effectively prevented, and thus the assembly efficiency is improved. With the method provided by the invention, the cloning steps are effectively reduced, the cloning efficiency is improved, and the assembling efficiency of the DNA fragments is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving DNA cloning and assembly efficiency by using short-chain primers to repress. Background technique [0002] With the popularization of whole genome sequencing technology and the development of DNA synthesis technology, synthetic biology has developed rapidly. In many research fields of synthetic biology, scientists need to synthesize large fragments of DNA, and the Saccharomyces cerevisiae genome synthesis project even needs to assemble DNA fragments into chromosomes. Therefore, exploring the rapid assembly method of large DNA fragments is a research hotspot today. [0003] Over the past few decades, restriction enzyme digestion-ligation has been the most commonly used cloning method. This DNA recombination technology began with the discovery of restriction enzymes and DNA ligases, and subsequently, various methods for ligating DNA molecules using ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/63C12N15/11
Inventor 戴俊彪林继伟秦怡然吴庆余何江海洋
Owner TSINGHUA UNIV
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