Recombinant DNA technology is one of the most important achievements in the development of modern molecular biotechnology. It is the core technology of Gene Engineering.
Recombinant DNA Technique is a method in which humans select a gene of interest (DNA fragment) to recombine with a gene carrier in vitro and transfer it to another cell or organism to improve and create new species and treat human diseases. .
The key to the development and application of this technology is to limit the discovery and application of enzymes.
First, the restriction enzyme
Restrictive endonucleases, also known as restriction enzymes. A nuclease ("molecular scalpel") that specifically cleaves a phosphodiester bond in a DNA strand.
Found in prokaryotes, more than 100 species have been isolated, and almost all prokaryotes contain this enzyme. It is an important class of tool enzymes in recombinant DNA technology and gene diagnosis.
1, the name of the restriction enzyme
Named according to its source. Such as:
EcoRI is derived from the RY13 strain of E. coli, and I refers to the first restriction enzyme isolated in this strain.
2. Restriction enzyme recognition sequence and cleavage formation
A typical 4-8 nucleotide sequence that each restriction enzyme recognizes and cleaves is referred to as a restriction site or a cut point.
Some commonly used restriction enzymes and cut points
Complementary end connection
There are three ways to generate different segments of the protruding end of the same sequence:
1) cutting with the same restriction enzyme;
2) cutting with different restriction enzymes that recognize the same target sequence;
3) Cleavage with restriction enzymes that recognize different target sequences but produce consistent cohesive ends.
3. Features:
Important uses in genetic engineering and genetic diagnosis based on the characteristics of the above restriction enzymes:
1) Regardless of the source of the DNA, the sticky ends produced by the same restriction enzyme are easily rejoined. Therefore, DNA of different species can be recombined. Such as human and plasmid DNA.
2) DNA analysis for the human genome with specific restriction sites.
3) Gene mutation changes the disappearance or new production of the cleavage site will change the length of the cleavage fragment. Applied to restriction fragment polymorphism analysis.
Second, the gene carrier and its choice
Vector: A tool for introducing foreign DNA of interest into a recipient cell and capable of self-replication and proliferation.
The carrier has the following characteristics:
1) The molecular weight is small, it is convenient to carry a large DNA fragment, and can enter and proliferate in the host cell;
2) There are a variety of restriction enzyme sites, and each restriction enzyme is preferably only a single point;
3) The vector after cleavage does not affect its ability to replicate after insertion of foreign DNA, and has a selectable marker gene (eg, a drug resistance gene).
Commonly used vectors are: plasmid, lambda phage, cosmid, BAC, YAC, PI, and the like.
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