The most basic technology of molecular biology is the expression and purification of protein. Firstly, the DNA sequence encoding the target protein was cloned (using PCR technology and restriction endonuclease) into a plasmid as an expression vector. Then the constructed plasmid is introduced into the host cell. The coding sequence is expressed by the expression system of the host cell, which is driven by a special promoter element on the plasmid. Plasmids usually carry antibiotic resistance labels to facilitate plasmid screening.
Plasmids can be inserted into bacterial or animal cells. Introducing foreign DNA into bacteria is called transformation, which can be achieved by electroporation, microinjection, positive absorption, fusion and other methods. Introducing foreign DNA into eukaryotic cells, such as animal cells, is called transfection. Transfection techniques include calcium phosphate method, liposome method and some patented commercial transfection reagents. DNA can also be carried into host cells by viruses or pathogens. When this virus or pathogen transfection technique is applied to cells, the term is "transducing cells".
Polymerase chain reaction (PCR) is a very common technique for replicating DNA in vitro. In short, PCR technology can make single-stranded DNA be copied millions of times, and the copied DNA sequence can also be modified in a predetermined way. For example, PCR technology can be used to introduce restriction sites or mutate (change) specific DNA bases. PCR technology can also obtain specific DNA fragments from c DNA library, or judge whether a cDNA library contains specific DNA fragments from another angle.
Gel electrophoresis is the most important technology in molecular biology. The basic principle is that DNA, RNA and protein can be separated by electric field. In agarose gel electrophoresis, DNA and RNA can be separated by agarose gel according to their molecular size. Similarly, protein can be separated by SDS-PAGE. In addition, due to different charges, protein can also be separated by isoelectric focusing electrophoresis.