This type of DNA cloning procedure involves a gene or other DNA fragment of interest is inserted into a circular piece of DNA called a plasmid. This combination creates a recombinant DNA in the bacteria, which has the means to produce proteins needed in the organism’s body. An example of this is the human insulin gene being expressed in E. coli bacteria to make insulin used by diabetics.

 In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources. The plasmid is then introduced to the bacteria. The bacteria then grows up and reproduces, passing the recombinant DNA to their offspring. The bacteria are used as “factories” to make the protein.

Ex: For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics.

-How?

1.Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).

 -The restriction enzyme recognizes a specific target sequence and cuts DNA into two pieces at or near that site, and the DNA ligase seals gaps in the DNA backbone.

2. Introduce or transform plasmid to the bacteria

-Plasmids and other DNA can be introduced into bacteria in transformation. During transformation, specially prepared bacterial cells are given a shock (such as high temperature) that encourages them to take up foreign DNA.

-A plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic. Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid will die, while bacteria carrying a plasmid can live and reproduce. Each surviving bacterium will give rise to a small, dot-like group, or colony, of identical bacteria that all carry the same plasmid.

3. The bacteria grows, reproduces, and passes the recombinant DNA to their offspring. Then the bacteria are used as “factories” to make protein. 

-For example, if the plasmid contained the human insulin gene, the bacteria would start transcribing the gene and translating the mRNA to produce many molecules of human insulin protein.

-Because there are other macromolecules and proteins floating around in the bacteria, the specific protein (in this case, insulin) must be purified in order to be expelled from the cell and used in other parts organism’s body. 

4. Research and Application

-Why?
 In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as “factories” to make the protein

EXAMPLES
-Gene analysis. In basic research labs, biologists often use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function.
- Gene therapy. In some genetic disorders, patients lack the functional form of a particular gene. Gene therapy attempts to provide a normal copy of the gene to the cells of a patient’s body. For example, DNA cloning was used to build plasmids containing a normal version of the gene that's nonfunctional in cystic fibrosis. When the plasmids were delivered to the lungs of cystic fibrosis patients, lung function deteriorated less quickly
-Biopharmaceuticals. DNA cloning can be used to make human proteins with biomedical applications, such as the insulin mentioned above. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.

How? -Nuclear transfer starts by removing the nucleus from one cell, such as a skin cell, and injecting the skin cell nucleus into another cell, such as a muscle cell, to clone the muscle cell into the skin cell. A new clone—a genetic copy of the donor—forms when the egg starts to divide. 

Why?- This technique inspired the development of SCNT (Somatic Cell Nuclear Transfer), which has become an important technique in stem cell research and in understanding the cellular mechanisms controlling embryonic development and nuclear reprogramming. Somatic cell nuclear transfer research is an important subset of stem cell research and could allow researchers to develop stem cell therapies that are specifically tailored to an individual’s medical condition and that do not trigger an immune rejection response.

Why?
PCR produces enough DNA to be analyzed using other techniques
-Gel electrophoresis
-Sequencing
-DNA cloning

PCR has practical applications
-Amplify genes associated with genetic disorders from the DNA of patients
-Test for bacterium or DNA virus in a patient's body