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A method for isothermally unraveling double-stranded DNA and preparing single-stranded DNA

A DNA probe and single-strand technology, applied in the field of molecular biology, can solve the problems of inability to get rid of thermal denaturation and heating equipment, reduce versatility, etc., achieve novel and simple preparation strategies, and expand the application range

Active Publication Date: 2020-05-08
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, both methods (1) and (2) cannot get rid of thermal denaturation and heating equipment, which violates the original intention of isothermal reaction; although method (3) can avoid dependence on instruments, endonucleases need to recognize specific sequences, which greatly reduces its versatility

Method used

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  • A method for isothermally unraveling double-stranded DNA and preparing single-stranded DNA
  • A method for isothermally unraveling double-stranded DNA and preparing single-stranded DNA
  • A method for isothermally unraveling double-stranded DNA and preparing single-stranded DNA

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Embodiment 1

[0036]Example 1. Isothermal unwinding of double-stranded DNA based on single-stranded DNA probe and recombinase

[0037] In order to facilitate the verification of the mechanism of isothermal unwinding of double-stranded DNA based on single-stranded DNA probe and recombinase through radioisotope labeling and polyacrylamide gel electrophoresis, the single-stranded DNA probe and double-stranded DNA are designed in this example to be the same length according to The technical solution for isothermal unwinding of double-stranded DNA of the present invention, the single-stranded DNA probe can directly and completely open the double-stranded DNA to pair with its complementary chain and replace another non-complementary chain, without the need for a single-stranded binding protein to stabilize the open-chain structure (single-stranded binding For the related research on the protein involved in stabilizing the open-chain structure, please refer to Examples 2-4, where the single-strande...

Embodiment 2

[0046] Example 2. Use group A single-stranded DNA probes (X1, X1f) to unwind the double-stranded template T12 (formed by annealing T1, T2), and then combine with ligase to prepare single-stranded DNA through a ligation reaction.

[0047] For the procedure of unwinding the double-stranded template to prepare single-stranded DNA by ligation, see figure 2 . Design two single-stranded DNA probes (X1, X1f), the 5' end of X1f is phosphorylated, while the 3' end of X1 is a hydroxyl group, and the 5' end is isotope P 32 mark.

[0048] In the presence of ATP or dATP, the recombinase RecA can form a complex with the single-stranded DNA probes X1 and X1f, respectively, and this complex can scan the double-stranded T12. When the region homologous to X1 and X1f is scanned, the double-stranded T12 is unwound, and X1 and X1f are respectively paired with the complementary strands and form a gap in the middle, X1 is at the 5' end, and X1f is at the 3' end. The other displaced strand binds ...

Embodiment 3

[0061] Example 3. Use group B single-stranded DNA probes (X1, X1r) to unwind double-stranded template T12 (formed by annealing T1, T2), and then combine with polymerase to prepare single-stranded DNA through extension reaction.

[0062] For the procedure of unwinding the double-stranded template to prepare single-stranded DNA by extension reaction, see image 3 . Design two single-stranded DNA probes (X1, X1r), with the isotope P at the 5' end of X1 32 Tag, paired complementary to the 3' downstream region of the T1 strand in the double-stranded template. X1r pairs complementary to the 3' downstream region of the T2 strand in the double-stranded template.

[0063] In the presence of ATP or dATP, the recombinase RecA can form a complex with the single-stranded DNA probes X1 and X1r, respectively, and this complex can scan the double-stranded T12. When the homologous sequence with X1 and X1r is scanned, the double-stranded T12 is opened in the corresponding region, X1 and X1r ...

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Abstract

The invention belongs to the technical field of molecular biology and provides a method for isothermally unwinding double-stranded DNA and a method for preparing single-stranded DNA based on the method. The technical solution for isothermal unraveling of double-stranded DNA is to use recombinase to bind single-stranded DNA probes to form a complex, which recognizes sequences homologous to single-stranded DNA probes in double-stranded DNA and invades double-stranded DNA, thereby achieving double-stranded DNA. The isothermal opening of the DNA, followed by the complementary pairing of the single-stranded DNA probe and one of the double strands, and the binding of the single-strand binding protein to the other strand stabilizes the open-strand structure. Based on this scheme and the characteristics that the 3'-end of the single-stranded DNA probe can be recognized by enzymes such as ligase and polymerase, the present invention also proposes a scheme for preparing single-stranded DNA, and introduces in detail the preparation of single-stranded DNA by ligase or elongase. Stranded DNA scheme. The above technical solutions can be completed under isothermal conditions without the need for an ATP cycle system, and even ATP can be replaced by dATP. The enzymes used are widely available and easy to obtain, providing a general platform for isothermal technology to use double-stranded DNA as the research object.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, relates to the unwinding of double-stranded DNA and the preparation of single-stranded DNA, and in particular relates to a universal method for isothermal unwinding of double-stranded DNA by using recombinase and single-stranded DNA and the preparation of single-stranded DNA using the same. method of stranding DNA. Background technique [0002] DNA is the carrier of genes, and the genetic information it carries encodes the amino acid sequences of all structural and functional proteins in the cell. With the exception of a small number of viruses, the genomes of organisms are almost always in the form of double strands. However, many basic operations based on DNA in vitro, such as DNA hybridization, ligation, amplification, etc., all utilize the principle of complementary base pairing between probes or primers and target DNA sequences. Therefore, the unwinding of double-stranded DNA is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/10C12P19/34C12Q1/6844C12Q2521/507C12Q2522/101C12Q2527/101C12Q2521/101C12Q2531/119
Inventor 唐卓陈刚毅
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S