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STEPS INVOLVED IN RECOMBINANT DNA TECHNOLOGY

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Racheal Smith

on 11 January 2015

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Transcript of STEPS INVOLVED IN RECOMBINANT DNA TECHNOLOGY

ISOLATION
The first step in making recombinant DNA is to isolate donor and vector DNA. . The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA


Insertion
A vector is any DNA molecule which is capable of multiplying inside the host to which our gene of interest is integrated for cloning. In this process restriction enzyme function as scissors for cutting the DNA molecules. Ligase enzyme is the joining enzyme that joins the vector DNA with the gene of interest. this will produce the recombinant DNA.
RECOMBINANT DNA TECHNOLOGY
Using Recombinant DNA technology, we can isolate and clone single copy of a gene or a DNA segment into an indefinite number of copies, all identical. These new combinations of genetic materials or Recombinant DNA '(rDNA)' molecules are introduced into host cells, where they propagate and multiply. The technique or methodology is called Recombinant DNA technology or "Genetic engineering".
Examples of Use of Recombinant DNA Technology
Insulin
Production
The DNA for insulin is first isolated.
A plasmid made of DNA is removed from a bacterial cell.
A restriction enzyme cuts the plasmid DNA open, leaving sticky ends.
The insulin gene, with complementary sticky ends is added.

Production of Insulin
STEPS INVOLVED IN RECOMBINANT DNA TECHNOLOGY
Ultracentrifugation
.A protocol for extracting plasmid DNA can be achieved by ultracentrifugation. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by punching a hole in the plastic centrifuge tube.
Alkaline Lysis
Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution. Phages such as λ also can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate.
WHAT IS RECOMBINANT DNA?
This is DNA that has been formed artificially by combining constituents from different organisms.
OBTAINING rDNA
Step 1
: The DNA fragment containing the gene sequence to be cloned (also known as ('insert') is isolated.
Step 2
: Cutting DNA
Step 3
: Joining DNA
Step 2
: ) Insertion of these DNA fragments into a host cell using a "vector" (carrier DNA molecule).

Step 3
: The rDNA molecules are generated when the vector self replicates in the host cell.
Step 4
: Transfer of the rDNA molecules into an appropriate host cell.
Step 5:
) Selection of the host cells carrying the rDNA molecule using a marker
Step 6
: Replication of the cells carrying rDNA molecules to get a genetically identical cells population or clone.

Choosing a Gene Cloning Vector
Recombinant DNA inserted
in cell.
Region of interest
is isolated
Both gene and
plasmid are joined.
Cell with
new DNA
is cloned.
Introducing Vector DNA into Host Cell
Plasmid Vectors
the vector is added to a flask containing a culture of E. coli
calcium ions usually in the form of calcium chloride are added to the flask, followed by a brief heat shock.
this allows holes to briefly appear in the cell surface membrane of the E. coli making it permeable to DNA and allowing the plasmids to enter
Introducing Vector DNA Into Host Cell
Phage Vectors:
Introduced by infection of bacterial lawn growing on an agar plate.
the culture or growth of viruses is made more difficult than the culture of bacteria or fungi by the fact that viruses will only grow
Placing the Gene in the Vector
Plasmid DNA
DNA molecules are small and can be easily separated based on size.
bacterial cells are broken open and chromosomal DNA is centrifuged down.
this leaves the plasmid DNA in the liquid above the pellet.
the plasmids are purified before cutting with a restriction enzyme.
restriction fragments from donor DNA are mixed with plasmid DNA and joined by their sticky ends. the initial attraction is due to the hydrogen bonds, but the sugar phosphate backbone is then joined using and enzyme called DNA ligase.
DNA ligase enzyme splices (joins) together the plasmid DNA and the Insulin DNA.
The plasmid (now genetically modified) is inserted back into the bacterium.
The bacterium host cell, divides and produces copies of the plasmid?
The Bacterium makes human insulin using the gene in the plasmid.
The insulin is extracted from the bacterial culture.
Examples of Use of Recombinant DNA Technology
Production of Insulin
Cutting DNA
The restriction enzyme
EcoRi
cuts a ircular DNA molecule bearing one target sequence, resulting in a linear molecule with single stranded sticky ends.
Joining DNA
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