DNA is also known as Genetic Engineering. The technique is to manually manipulate DNA molecules in vitro according to a certain purpose and protocol, recombine the same or heterologous genes, and then introduce them into appropriate recipient cells. As the cells multiply, DNA recombinants Amplification is obtained and expression is obtained at the same time. This allows a large amount of recombinant DNA to be obtained. Recombinant DNA technology is also known as molecular cloning (Molecular Cloning). The term clone is intended to mean the sum of the offspring of an individual through asexual reproduction. Molecular cloning is the introduction of a single gene into an appropriate recipient cell to replicate it, resulting in a large number of copies of the recombinant DNA.
(1) Tool enzymes commonly used in recombinant DNA
The DNA is cleaved into small fragments in vitro and then recombined, which requires the participation of a series of enzymes. These enzymes are called tool enzymes. The commonly used tool enzymes are the following.
l. Restriction Endonuclease An enzyme that recognizes and cleaves a specific nucleotide sequence within a double-stranded DNA molecule is called a restriction endonuclease. Restriction endonucleases are found in prokaryotes, and their natural biological function is the defense mechanism that constitutes bacteria against foreign invading DNA. Restriction endonucleases are often divided into three types: Type I restriction endonucleases differ in the recognition site of the DNA strand from the cleavage site and cannot produce specific DNA fragments; Type II restriction endonuclease Identify and cleave the same specific nucleotide sequence on the DNA strand to produce a specific DNA fragment; the type III restriction endonuclease has a specific cleavage site, but it has multiple subunits and is different by Gene coding. Therefore, type I and type III endonucleases are of little significance as a tool enzyme, and the so-called restriction endonuclease is a type II enzyme. Approximately 400 restriction endonucleases have been isolated from more than 250 microorganisms, and the DNA sequences they recognize typically contain 4-6 nucleotides, some of which produce blunt ends and some produce sticky ends. Restriction endonucleases are the most important tool enzymes in gene manipulation.
2. DNA ligase An enzyme that splicing two DNA fragments is called DNA ligase. DNA ligase not only plays a role in DNA replication (such as the attachment of Okazaki fragments) and DNA repair, but is also used to link fragments of two DNAs in in vitro recombination of DNA.
3. Reverse transcriptase reverse transcriptase plays a role in the replication of RNA viruses. The molecular cloning technique utilizes the activity of this enzyme to reverse-transcribe mRNA into complementary DNA (cDNA), which is more stable than mRNA and easy to manipulate.
4. Other enzymes In the molecular cloning technology, according to the needs of experimental design, various other enzymes with different catalytic effects can be selected. Such as terminal transferase, DNA polymerase and the like.
(2) vectors commonly used in recombinant DNA
The Cloning Vector is another DNA molecule that carries a foreign gene (the gene of interest) into the recipient cell and replicates and expresses it in the recipient cell. The cloning vector has certain genetic properties such as drug resistance, plaque, and the like. The most commonly used carriers are the following:
1. Plasmid (Plasmid) is a small molecule DNA that can replicate autonomously by extrachromosomal bacteria, mostly circular double-stranded. The plasmid carries certain genetic traits, such as resistance to certain antibiotics. Receptor bacteria carrying the gene of interest can be selected based on the characteristics of the plasmid. Since the plasmid has the ability to self-replicate, the plasmid can be kept constant to the progeny cells during cell division, and the carried target gene can be inherited. The first commonly used plasmid was pBR322.
2. Phage Since phage can infect bacteria and replicate in bacteria, they can also serve as vectors for cloning. Commonly used phage such as lambda DNA.
3. Viral viruses can infect eukaryotes and replicate in host cells. It can be used as a carrier after proper modification. The commonly used SV40 is a monkey virus.
4. Yeast Artificial Chromosome (YAC) This is a vector artificially spliced ​​by the yeast gene and plasmid pBR322. This vector can carry large fragments of DNA (102 to 103 kb) and can replicate in E. coli with the basic functional units of the yeast genome. It is a useful tool in human genome research.
(3) The basic process of genetic engineering
Genetic engineering can be roughly divided into the following steps: preparation of the target gene, selection and preparation of the vector DNA, recombination of the target gene and the vector DNA, introduction of the recombinant DNA molecule into the recipient cell, screening and identification of the recipient cell containing the recombinant molecule, Amplification of expression and identification of recombinant molecules and genes of interest.
1. The common method for obtaining the target gene of the target gene includes: 1 by restriction enzyme digestion into chromosomal DNA into a desired DNA fragment; 2 reverse transcription reaction, using mRNA as a template, and synthesizing complementary DNA from mRNA under the action of reverse transcriptase That is, cDNA; 3 shorter nucleotide sequence can be artificially synthesized by using an automatic DNA synthesizer according to the nucleotide sequence encoded by the amino acid in the known polypeptide chain; 4, using the PCR technique, in vitro amplification of the desired gene of interest.
2. Selection and preparation of vector DNA Different vectors were selected depending on the nature of the gene of interest, and the vector was purified and treated with restriction enzymes.
3. Recombination of the gene of interest and the vector DNA in vitro The DNA of interest and the vector DNA are ligated into recombinant DNA in vitro using DNA ligase.
4. Introduction of Recombinant DNA into Receptor Cells Recombinant DNA can be introduced into recipient cells by physical and chemical methods and expressed in the recipient cells. The process by which a recombinant plasmid DNA molecule is introduced into a recipient cell is called transformation, and the process by which the recombinant phage and the virus are introduced into the recipient cell is called transfection.
5. Screening and Identification The genetic markers of the vector DNA and certain characteristics of the gene of interest are used to screen for recipient cells containing the recombinant and to identify recombinants.
6. Amplification of recombinant and target gene expression Recombinant DNA molecules can replicate autonomously in recipient cells, amplify the target gene, and transcribe and translate, express and secrete biologically active proteins, which is needed Genetic engineering products.
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