Molecular biology: A discipline that studies the material basis of life phenomena at the molecular level. Study the physical and chemical properties and changes of cellular components and the relationship between these properties and changes and life phenomena, such as the transmission of genetic information, gene structure, replication, transcription, translation, expression regulation and physiological functions of expression products, as well as cell signals transduction, etc.
The most basic technology in molecular biology is protein expression and purification. First, the DNA sequence encoding the protein of interest is cloned (using PCR technology and restriction enzymes) into a plasmid serving as an expression vector. The constructed plasmid is then introduced into the host cell. The coding sequence is expressed by the host cell expression system driven by special promoter elements on the plasmid. Plasmids often also carry antibiotic resistance tags to facilitate plasmid screening.
Plasmids can be inserted into bacterial or animal cells. The introduction of exogenous DNA into bacteria is called transformation, which can be achieved through electroporation, microinjection, positive absorption, and fusion; the introduction of exogenous DNA into eukaryotic cells, such as animal cells, is called transfection, and transfection technology Including calcium phosphate method, liposome method and some patented commercial transfection reagents. DNA can also be introduced into host cells using viruses or pathogenic bacteria as vectors; when applying this viral or pathogenic transfection technology to cells, the terminology is “transduce” the cells.
Polymerase chain reaction (PCR) is an extremely versatile technique for replicating DNA in vitro. In short, PCR technology allows single-stranded DNA to be copied millions of times, and it also allows the copied DNA sequence to be modified in a predetermined way. For example, PCR technology can be used to introduce restriction enzyme sites, or to mutate (change) specific DNA bases. PCR technology can also be used to obtain specific DNA fragments from cDNA libraries, or from another perspective, to determine whether a cDNA library contains specific DNA fragments.
Gel electrophoresis is the most important technology in molecular biology. The basic principle is that DNA, RNA and proteins can be separated using electric fields. In agarose gel electrophoresis, DNA and RNA can be separated by agarose gel according to their molecular size. Similarly, proteins can be separated by molecular weight by SDS-polyacrylamide gel electrophoresis (SDS-PAGE); in addition, proteins can also be separated by isoelectric focusing electrophoresis due to differences in charges.