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What is Recombinant DNA?
DNA recombination is the process of re-covalent combination of genetic information that occurs within or between DNA molecules. It includes intra-genomic large segment DNA translocation, inter-genomic large segment DNA transfer and even genomic integration. Among them, intragenomic large DNA translocation is also known as DNA rearrangement. DNA recombination exists in all kinds of organisms.In eukaryotes, it mainly occurs in the homologous chromosome exchange of meiosis, while the genomes of bacteria and phage are haploid, and their DNA can be recombined in a variety of ways.
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Recombinant DNA Technology
Recombinant DNA technology, also known as genetic engineering or molecular cloning, is the process of combining DNA fragments from one organism with DNA from another in vitro to form a new DNA molecule, which is introduced into the host cell to stabilize its existence and expression. This technology is at the heart of modern biotechnology and has revolutionized a number of fields, including the study of gene function, protein production, genetic improvement and disease treatment. The vectors used in recombinant DNA technology are mainly plasmids and mild phages, and almost all vectors in practical applications are modified plasmids or mild phages. The British microbial geneticist W. Hayes and the American microbial geneticist, J. Lederberg, etc. firstly recognized that the F factor of E. coli was an extrachromosomal genetic factor in 1952, and in 1953, the French scholars, such as P. Frédéric, etc., found that E. coli produces colistin as a trait controlled by an extrachromosomal colistin factor. 1957, the Japanese scholars discovered the resistance plasmid. plasmids. The latter two types of plasmids are widely used in genetic engineering. Recombinant DNA technology stems from basic theoretical research in two areas - restriction endonucleases and gene vectors.
Recombinant DNA Technology.
What is the Method of Recombinant DNA?
The more well-studied modes of DNA recombination are homologous recombination, site-specific recombination and transposition. Homologous recombination refers to recombination between two homologous chromosomes with identical or similar gene sequences. Site-specific recombination refers to recombination at a specific site, usually involving a particular DNA sequence. Transposition, on the other hand, is the movement of a DNA fragment from one location to another.
Mechanism of Recombinant DNA
Plasmids have the advantages of stability, reliability and ease of handling. For cloning small and simple DNA fragments (less than or equal to 10 kb), plasmids are better than any other vector. Cloning on a plasmid vector is simple in principle. First, the plasmid DNA and the target DNA fragments are cut by restriction endonuclease, then the two are ligated in vitro, and then the resulting recombinant plasmid is transferred to the bacteria, and the process is completed. However, in practice, it is difficult to distinguish the recombinant plasmid with inserted exogenous DNA from the vector molecule without insertion and self-cyclization. By adjusting the ratio of the concentration of exogenous DNA fragments and vector DNA in the ligation reaction, the auto-cyclization of vectors can be limited to a certain extent, and some special cloning strategies, such as dephosphorylation of vectors, can be adopted to minimize the auto-cyclization of vectors, and genetic means, such as the a-complementation phenomenon, can be used to identify recombinants and non-recombinants.
Process of Recombinant DNA
Acquisition and Processing of DNA Fragments
DNA fragments are amplified and enzymatically cleaved by PCR. Restriction endonucleases are used to recognize and cleave specific DNA sequences to produce complementary sticky or flat ends, and DNA ligases can join these fragments to form recombinant DNA molecules.
Vector Construction
Recombinant DNA molecules are formed in vitro by ligating the target gene fragments to vector DNA. Commonly used vectors include plasmids, phages, viral vectors, etc. These vectors have the ability to self-replicate and carry selection marker genes, which are easy to screen and maintain in the host cell.
Ligation of DNA Fragments and Plasmid Vector
There are several strategies for ligation reactions of exogenous DNA fragments and plasmid vectors:
1. With non-complementary protruding ends
Digestion with two different restriction endonucleases can produce DNA fragments with non-complementary sticky ends, which are also the easiest to clone. In general, commonly used plasmid vectors have multiple cloning sites composed of different restriction enzyme recognition sequences, so it is almost always possible to find vectors with restriction enzyme sites that match the exogenous DNA fragment ends, and thus to clone the exogenous fragment into the vector in a targeted manner. It is also possible to artificially attach different cleavage sites to the ends of the DNA fragments during PCR amplification.
2. With identical sticky ends
Such ends can be obtained by treatment with the same enzyme or homologous enzyme. Since the plasmid vector must also be digested with the same enzyme to obtain the same two identical sticky ends, both the exogenous fragment and the plasmid vector DNA may be cyclized or several molecules may be linked together to form oligomers in the ligation reaction, and both directions of ligation are possible. Therefore, the concentration of both DNAs in the ligation reaction must be carefully adjusted to maximize the number of correct ligation products. The 5' phosphate group of the vector DNA can also be removed by alkaline phosphatase to maximize the inhibition of self-cyclization of the plasmid DNA. Exogenous DNA fragments with 5'phosphate can be effectively ligated to the dephosphorylated vector, resulting in a ring-opening molecule with two nicks, which can be repaired automatically during the amplification process after being transferred into the E. coli receptor bacteria.
3. With flat ends
The fragments produced by the digestion of restriction enzymes or nucleic acid exonucleases that produce flat ends, or by the DNA polymerase to make up the flat fragments Because the ligation efficiency of the flat ends is much lower than that of the sticky ends, the concentration of T4 DNA ligase and the concentration of the exogenous DNA and the vector DNA in the ligation reaction are much higher. Therefore, the concentration of T4 DNA ligase and the concentration of exogenous DNA and vector DNA are much higher in the ligation reaction. It is usually necessary to add a low concentration of polyethylene glycol to promote the cohesion of DNA molecules into aggregates in order to improve the conversion efficiency.
Transformation or Transfection
Introduction of recombinant DNA molecules into host cells.
Screening and Characterization
Identification of recombinant cells by antibiotic resistance screening, blue and white spot screening, PCR validation, and zymography analysis.
Physiological Significance of Recombinant DNA
DNA recombination plays a variety of important roles in living organisms and has the following physiological significance:
- Participation in DNA replication: DNA recombination plays a key role in the DNA replication process, ensuring the accurate transmission of genetic information.
- Involved in DNA repair: DNA recombination is involved in the repair of double-strand breaks, helping to repair damaged DNA molecules and maintain genetic stability.
- Participate in gene expression regulation DNA recombination can affect the level and regulation of gene expression by changing the arrangement of gene sequences.
- Promote correct chromosome segregation: During eukaryotic cell division, DNA recombination helps to ensure the correct segregation of chromosomes and avoid the loss of genetic information.
- Generate genetic diversity: DNA recombination produces new genetic combinations by recombining gene sequences, providing the raw material for species evolution.
- Key role in antibody synthesis: DNA recombination plays an important role in the rearrangement of antibody genes, generating diverse antibody combinations and enhancing the immune ability of organisms.
- Realization of programmed gene rearrangement: During development, DNA recombination participates in programmed gene rearrangement and regulates the normal development of embryos.
- Participate in viral genome integration: DNA recombination plays a key role in viral infection of host cells, helping viruses to integrate into the host cell genome.
Applications of Recombinant DNA
Recombinant DNA has become an indispensable research object in modern biotechnology and is widely used in recombinant DNA technology, transgenic technology, gene targeting and gene therapy.
- Recombinant DNA Technology: Through recombinant DNA technology, DNA molecules containing specific gene sequences can be constructed for gene cloning, gene expression and protein production.
- Transgenic Technology: Recombinant DNA technology plays a key role in transgenic technology by introducing exogenous genes into target organisms to change their genetic characteristics.
- Gene Targeting: DNA recombinant technology can be used to create specific gene-targeting sequences for the study of gene function and regulatory mechanisms.
- Gene Therapy: DNA recombinant technology is used in gene therapy to repair and replace abnormal genes and treat hereditary diseases.Reference
* Only for research. Not suitable for any diagnostic or therapeutic use.
