Ever since the discovery of DNA restriction enzyme and DNA ligase, the DNA recombinant technology has developed rapidly, and has been widely used for determining the cause of diseases and potential cures. Nowadays it becomes the most basic part of studies involving essentially all aspects of biology.
Molecular cloning means isolate a piece of DNA and amplify it via recombinant DNA technology. The DNA source can be genomic DNA, cDNA, or PCR amplified DNA fragments. These DNA pieces are cut by restriction enzymes to create compatible DNA ends with the vectors. They are then grown in E. coli for amplification.
Library construction and screening
There are two types of libraries depending on the DNA source: genomic library and cDNA library. The desired clones can be screened using hybridization technique with a labeled probe.
Analysis of genes and transcripts
When a plasmid is digested by restriction enzymes, the length of each fragment can be analyzed on a gel, then the physical map of the plasmid can be constructed. The DNA on gel can be analyzed by hybridization after transfer onto a membrane, this is called Southern blot. A similar procedure called northern blot is used to detect mRNA on a membrane. Reverse transcription mediated PCR can also be used to analyze mRNA from cells.
Application of recombinant DNA technology
The recombinant DNA technology is the foundation of modern agriculture and biomedical science, its impact reaches every corner of the research, therapy and normal daily life. In agriculture, it is used to generate genetically modified crops for higher yield or better quality; in biomedical science, it is used to analyze biological process, analyze human genetic disease, and generate recombinant protein for research and drug development.