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The discovery of restriction enzymes, proteins that cut (or "digest") DNA in a sequence specific manner, has made it possible to take DNA from one organism and combine it with DNA from another. Scientists have harnessed this capability, using restriction enzymes as "molecular scissors" to cut DNA at specific points to isolate regions of interest. These DNA fragments can then be isolated and joined with other pieces of DNA by adding DNA ligase, an enzyme which creates a covalent between the backbones of the DNA fragments. In this process of "molecular cloning", isolated fragments of DNA are first inserted into a DNA vector carried in a host microbe such as a bacterium or virus. In bacteria, the DNA cloning vector is typically a plasmid, a circular, nonessential, extrachromosomal piece of DNA. In viruses the vector is the viral DNA itself. The cloning of relatively small DNA fragments (less than several thousand base pairs) is typically accomplished using plasmid vectors in bacteria. Larger DNA fragments require viral vectors or vectors called artificial chromosomes that are used in yeast or bacterial hosts. Once ligated together, these recombinant DNA molecules (vector + insert) can then be inserted into the appropriate host (e.g. bacteria for plasmids). The host containing the recombinant vector can be cultured to produce essentially unlimited amounts of DNA containing the fragment of interest. These recombinant DNA molecules can be used for a wide variety of applications. For example, they can be used to: • study of the cloned DNA (often a gene) to learn about its function • produce genetically modified organisms (GMOs) • produce recombinant proteins by expressing the cloned DNA in a host cell (e.g. insulin for diabetics) Traditional molecular cloning involves the following general steps: 1) Preparation of the Vector and Insert – digesting the vector and insert with restriction enzymes that will make ligating them together possible. The choice of restriction enzymes will depend on the cut sites available in both vector and insert and the strategy that will be employed. 2) LIGATION – combining the DNA fragment of interest with the plasmid vector using DNA ligase 3) TRANSFORMATION – introduction of the recombinant vector into a bacterial host 4) SCREENING – identifying bacterial clones carrying the DNA of interest Preparation of the vector and insert requires an essential working knowledge of the plasmid to be used. Plasmids usually have several essential features that make them useful: • origin of replication (ori) - a DNA sequence that directs replication of the plasmid DNA. Without it the plasmid would never be copied by DNA replication in the bacterial host and thus never passed on to daughter cells. • Selectable marker – a gene that ensures that allows identification of cells that have been transformed with the plasmid. For example, the ampicillin resistance gene (bla) encodes an enzyme that degrades the antibiotic ampicillin. This ensures that only bacteria containing the plasmid will survive exposure to ampicillin which is extremely useful in the screening process. • multiple cloning site (MCS) - a short stretch of DNA containing a variety of unique restriction sites (they only cut the plasmid once (within the MCS). This is where a DNA fragment of interest will be inserted into the plasmid.

Once ligated together, these recombinant DNA molecules (vector + insert) can then be inserted into the appropriate host (e.g. bacteria for plasmids). The host containing the recombinant vector can be cultured to produce essentially unlimited amounts of DNA containing the fragment of interest. These recombinant DNA molecules can be used for a wide variety of applications. For example, they can be used to:
• study of the cloned DNA (often a gene) to learn about its function
• produce genetically modified organisms (GMOs)
• produce recombinant proteins by expressing the cloned DNA in a host cell (e.g. insulin for diabetics)

Traditional molecular cloning involves the following general steps:

1) Preparation of the Vector and Insert – digesting the vector and insert with restriction enzymes that will make ligating them together possible. The choice of restriction enzymes will depend on the cut sites available in both vector and insert and the strategy that will be employed.

2) LIGATION – combining the DNA fragment of interest with the plasmid vector using DNA ligase

3) TRANSFORMATION – introduction of the recombinant vector into a bacterial host

4) SCREENING – identifying bacterial clones carrying the DNA of interest

Preparation of the vector and insert requires an essential working knowledge of the plasmid to be used. Plasmids usually have several essential features that make them useful:
• origin of replication (ori) - a DNA sequence that directs replication of the plasmid DNA. Without it the plasmid would never be copied by DNA replication in the bacterial host and thus never passed on to daughter cells.
• Selectable marker – a gene that ensures that allows identification of cells that have been transformed with the plasmid. For example, the ampicillin resistance gene (bla) encodes an enzyme that degrades the antibiotic ampicillin. This ensures that only bacteria containing the plasmid will survive exposure to ampicillin which is extremely useful in the screening process.
• multiple cloning site (MCS) - a short stretch of DNA containing a variety of unique restriction sites (they only cut the plasmid once (within the MCS). This is where a DNA fragment of interest will be inserted into the plasmid.

The discovery of restriction enzymes, proteins that cut (or "digest") DNA in a sequence specific manner, has made it possible to take DNA from one organism and combine it with DNA from another. Scientists have harnessed this capability, using restriction enzymes as "molecular scissors" to cut DNA at specific points to isolate regions of interest. These DNA fragments can then be isolated and joined with other pieces of DNA by adding DNA ligase, an enzyme which creates a covalent between the backbones of the DNA fragments. In this process of "molecular cloning", isolated fragments of DNA are first inserted into a DNA vector carried in a host microbe such as a bacterium or virus. In bacteria, the DNA cloning vector is typically a plasmid, a circular, nonessential, extrachromosomal piece of DNA. In viruses the vector is the viral DNA itself. The cloning of relatively small DNA fragments (less than several thousand base pairs) is typically accomplished using plasmid vectors in bacteria. Larger DNA fragments require viral vectors or vectors called artificial chromosomes that are used in yeast or bacterial hosts.

Once ligated together, these recombinant DNA molecules (vector + insert) can then be inserted into the appropriate host (e.g. bacteria for plasmids). The host containing the recombinant vector can be cultured to produce essentially unlimited amounts of DNA containing the fragment of interest. These recombinant DNA molecules can be used for a wide variety of applications. For example, they can be used to:
• study of the cloned DNA (often a gene) to learn about its function
• produce genetically modified organisms (GMOs)
• produce recombinant proteins by expressing the cloned DNA in a host cell (e.g. insulin for diabetics)

Traditional molecular cloning involves the following general steps:

1) Preparation of the Vector and Insert – digesting the vector and insert with restriction enzymes that will make ligating them together possible. The choice of restriction enzymes will depend on the cut sites available in both vector and insert and the strategy that will be employed.

2) LIGATION – combining the DNA fragment of interest with the plasmid vector using DNA ligase

3) TRANSFORMATION – introduction of the recombinant vector into a bacterial host

4) SCREENING – identifying bacterial clones carrying the DNA of interest

Preparation of the vector and insert requires an essential working knowledge of the plasmid to be used. Plasmids usually have several essential features that make them useful:
• origin of replication (ori) - a DNA sequence that directs replication of the plasmid DNA. Without it the plasmid would never be copied by DNA replication in the bacterial host and thus never passed on to daughter cells.
• Selectable marker – a gene that ensures that allows identification of cells that have been transformed with the plasmid. For example, the ampicillin resistance gene (bla) encodes an enzyme that degrades the antibiotic ampicillin. This ensures that only bacteria containing the plasmid will survive exposure to ampicillin which is extremely useful in the screening process.
• multiple cloning site (MCS) - a short stretch of DNA containing a variety of unique restriction sites (they only cut the plasmid once (within the MCS). This is where a DNA fragment of interest will be inserted into the plasmid.

Added by Jacob J.

Biology for AP Courses

Julianne Zedalis, John Eggebrecht

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01/12/2022

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The discovery of restriction enzymes, proteins that cut (or "digest") DNA in a sequence specific manner, has made it possible to take DNA from one organism and combine it with DNA from another. Scientists have harnessed this capability, using restriction enzymes as "molecular scissors" to cut DNA at specific points to isolate regions of interest. These DNA fragments can then be isolated and joined with other pieces of DNA by adding DNA ligase, an enzyme which creates a covalent between the backbones of the DNA fragments. In this process of "molecular cloning", isolated fragments of DNA are first inserted into a DNA vector carried in a host microbe such as a bacterium or virus. In bacteria, the DNA cloning vector is typically a plasmid, a circular, nonessential, extrachromosomal piece of DNA. In viruses the vector is the viral DNA itself. The cloning of relatively small DNA fragments (less than several thousand base pairs) is typically accomplished using plasmid vectors in bacteria. Larger DNA fragments require viral vectors or vectors called artificial chromosomes that are used in yeast or bacterial hosts. Once ligated together, these recombinant DNA molecules (vector + insert) can then be inserted into the appropriate host (e.g. bacteria for plasmids). The host containing the recombinant vector can be cultured to produce essentially unlimited amounts of DNA containing the fragment of interest. These recombinant DNA molecules can be used for a wide variety of applications. For example, they can be used to: study of the cloned DNA (often a gene) to learn about its function produce genetically modified organisms (GMOs) produce recombinant proteins by expressing the cloned DNA in a host cell (e.g. insulin for diabetics) Traditional molecular cloning involves the following general steps: 1) Preparation of the Vector and Insert – digesting the vector and insert with restriction enzymes that will make ligating them together possible. The choice of restriction enzymes will depend on the cut sites available in both vector and insert and the strategy that will be employed. 2) LIGATION – combining the DNA fragment of interest with the plasmid vector using DNA ligase 3) TRANSFORMATION – introduction of the recombinant vector into a bacterial host 4) SCREENING – identifying bacterial clones carrying the DNA of interest Preparation of the vector and insert requires an essential working knowledge of the plasmid to be used. Plasmids usually have several essential features that make them useful: origin of replication (ori) - a DNA sequence that directs replication of the plasmid DNA. Without it the plasmid would never be copied by DNA replication in the bacterial host and thus never passed on to daughter cells. Selectable marker – a gene that ensures that allows identification of cells that have been transformed with the plasmid. For example, the ampicillin resistance gene (bla) encodes an enzyme that degrades the antibiotic ampicillin. This ensures that only bacteria containing the plasmid will survive exposure to ampicillin which is extremely useful in the screening process. multiple cloning site (MCS) - a short stretch of DNA containing a variety of unique restriction sites (they only cut the plasmid once (within the MCS). This is where a DNA fragment of interest will be inserted into the plasmid.