Molecular biology is developed on the basis of biochemistry to study the life cycle of nucleic acid and protein structure and function, and to study the mechanisms of pathogenesis, diagnosis, treatment and prognosis at the level of nucleic acid and protein molecules. Among them, genetic engineering (genetic technology, genetic recombination) is a hotspot in molecular biology research. These technologies can transform or amplify genes and gene products, so that a small number of research objects can reach the analytical level. It is a method for studying gene regulation and expression, and is also a molecule. Levels of research on disease development mechanisms, genetic diagnosis, and gene therapy. Transformation, transfection, transduction, translocation, etc. are the ways in which genetic recombination exists in nature, and are also commonly used in artificial genetic recombination. One of the purposes of gene recombination is gene cloning, which can be understood as a gene identical to the structure of a template molecule amplified by using a single gene as a template. The micro- and mixed target genes that need to be analyzed and studied are easy to purify and can be increased for analysis.

The process by which a foreign gene causes changes in the biological properties of a cell is called transformation, and the process of introducing a foreign gene into a bacterium by a phage is called transfection. The process of transferring a genetic material from one host to another using a vector (phage or virus) is called transduction. The process of transferring one or a group of genes from one place to another in the genome is called transposition, and these swimming genes are called transposons.

First, common tools for genetic engineering

(1) Carrier

Vector (Vector) is a tool for introducing, amplifying, and expressing foreign DNA (the gene of interest) into a host cell. The vector includes a plasmid, a phage, a single-stranded filamentous phage, a cohesive terminal plasmid (clay), a virus, and the like. The vector has self-replication; there are selectable genetic markers for screening and identification; there are sites for insertion of foreign DNA; and its own small size.

The plasmid is present in a variety of bacteria and is an independent genetic element other than the chromosome (nucleus). It consists of double-stranded circular DNA, almost completely naked, with few protein binding. The plasmid has a tight type and a relaxed type. The stringent type is replicated by DNA polymerase III, and one cell can replicate 1-5 plasmids. The relaxation type is replicated by DNA polymerase I, and one cell can replicate 30-50 plasmids. If chloramphenicol is used to prevent protein synthesis, the plasmid can effectively utilize the raw material and replicate more plasmids. The plasmid has been modified into a wide variety, commonly used pBR322, pUC series. These plasmids contain multiple basic genes, such as the replication initiation region (replication origin Ori) for easy replication and amplification; antibiotic resistance markers (anti-ampicillin Apr, anti-tetracycline Tcr, etc.) or Escherichia coli partial lactose operons (E. Coli LacZ), etc., facilitates the screening of genetic recombinants; gene mobilizers (lactose operon Lac, tryptophan operon Trp, etc.) and transcription termination sequences, facilitating the transcription and translation of foreign genes inserted. There are also many restriction sites for restriction endonucleases on the plasmid, namely gene insertion sites, also known as gene recombination sites, and gene cloning sites.

Commonly used phage vectors are the single-stranded phage M13 system; the double-stranded phage system. The phage should be used in conjunction with the corresponding host cell. The above carriers have their own characteristics, which are easy to select and flexible to apply.

(2) Tool enzyme

Tool enzymes are an indispensable tool for gene recombination technology. There are mainly restriction enzymes, ligases, nucleic acid polymerases, reverse transcriptases, nucleases and the like.

Restriction enzymes are classified as type I and type II restriction endonucleases. Type II rigorously recognizes nucleic acid sequences and cleaves DNA duplexes at specific nucleotides in the recognition region. Therefore, it is generally referred to as type II restriction endonuclease. The recognition is divided into four nucleotides and six nucleotides, the sequence of which is rotationally symmetric. The slit is separated from the end and the sticky end to produce 3'-OH and 5'-P ends. There are many endonuclease varieties, and the reaction conditions such as temperature and buffer amount (generally 1 μg DNA/2-5 unit enzyme) should be noted.