DNA cloning is the process of making multiple, identical copies of a particular piece of DNA.
DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene.
In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid.
The plasmid is introduced into bacteria via a process called transformation, and bacteria carrying the plasmid are selected using antibiotics.
Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express the gene and make protein.
BASIC STEPS OF GENE CLONING are :
1. Cutting and pasting DNA and Vectors
2. Bacterial transformation and selection
3.Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it
Basic Requirements for Gene Cloning:
Cloning Vectors
Isolation Procedure for DNA fragment and Vectors
Appropriate Enzyme for Purified DNA manipulation
Host Cells
Agar with Antibiotics
Vectors for Gene Cloning: Plasmid and Bacteriophage :
Characteristic of a Vector for Gene cloning
It must be able to replicate with in the host cells
It needs to be small , Ideally less than 10 kb in size
Two kind of DNA molecule that satisfy the above criteria can be found in bacterial cells, namely Plasmid and Bacteriophage chromosomes
Plasmid:Is a circular molecule of DNA that lead un independent existence in the bacteria cells.
Bacteriophages, or phages are viruses that specifically
infect bacteria.
Characteristic of a Vector for Gene cloning
It must be able to replicate with in the host cells
It needs to be small , Ideally less than 10 kb in size
Two kind of DNA molecule that satisfy the above criteria can be found in bacterial cells, namely Plasmid and Bacteriophage chromosomes
Plasmid:Is a circular molecule of DNA that lead un independent existence in the bacteria cells.
Bacteriophages, or phages are viruses that specifically
infect bacteria.
Characteristic of a Vector for Gene cloning
It must be able to replicate with in the host cells
It needs to be small , Ideally less than 10 kb in size
Two kind of DNA molecule that satisfy the above criteria can be found in bacterial cells, namely Plasmid and Bacteriophage chromosomes
Plasmid:Is a circular molecule of DNA that lead un independent existence in the bacteria cells.
Bacteriophages, or phages are viruses that specifically
infect bacteria.
The procedure for total DNA preparation from a culture of bacterial cells can be divided into four stages (Figure 3.1):
A culture of bacteria is grown and then harvested.
The cells are broken open to release their contents.
This cell extract is treated to remove all components except the DNA.
The resulting DNA solution is concentrated
Treatment with EDTA and lysozyme is carried out in the presence
of sucrose, which prevents the cells from bursting immediately.
2. What is Gene Cloning?
DNA cloning is the process of making multiple, identical
copies of a particular piece of DNA.
It is an insertion of DNA fragment to a vector molecule
and then transferring to the host cells( bacteria) .
3. BASIC STEPS OF GENE CLONING
The basic steps are:
1. Cutting and pasting DNA and Vectors
2. Bacterial transformation and selection
3.Grow up lots of plasmid-carrying bacteria
and use them as "factories" to make the
protein. Harvest the protein from the bacteria
and purify it
4. Basic Requirements for Gene Cloning
Cloning Vectors
Isolation Procedure for DNA fragment and
Vectors
Appropriate Enzyme for Purified DNA
manipulation
Host Cells
Agar with Antibiotics
5. Vectors for Gene Cloning: Plasmid and Bacteriophage
Characteristic of a Vector for Gene cloning
It must be able to replicate with in the host cells
It needs to be small , Ideally less than 10 kb in size
Two kind of DNA molecule that satisfy the above criteria can be
found in bacterial cells, namely Plasmid and Bacteriophage
chromosomes
Plasmid:Is a circular molecule of DNA that lead un independent
existence in the bacteria cells.
Bacteriophages, or phages are viruses that specifically
infect bacteria.
9. 1. Purification of fragment DNA and vectors
The procedure for total DNA preparation from a culture of
bacterial cells can be divided into four stages (Figure 3.1):
1) A culture of bacteria is grown and then harvested.
2) The cells are broken open to release their contents.
3) This cell extract is treated to remove all components
except the DNA.
4) The resulting DNA solution is concentrated.
18. 2. Manipulation of purified DNA
DNA manipulative enzymes can be grouped into
four broad classes, depending on thet ype of
reaction that they catalyse:
1. Nucleases are enzymes that cut, shorten, or
degrade nucleic acid molecules.
2. Ligases join nucleic acid molecules together.
3. Polymerases make copies of molecules.
4. Modifying enzymes remove or add chemical
groups.
19. Nuclease
Nucleases degrade DNA molecules by breaking the phosphodiester
bonds that link one nucleotide to the next in a DNA strand.
There are two different kinds of nuclease
1. Exonucleases remove nucleotides one at a time from the end of a
DNA molecule.
Bal31
Exonulease III
2. Endonucleases are able to break internal phosphodiester bonds
within a DNA molecule.
S1 nuclease
DNease
Restriction Endonuclease
37. Uses of DNA cloning
DNA molecules built through cloning techniques are
used for many purposes in molecular biology.
A short list of examples includes:
Biopharmaceuticals
Gene therapy
Gene analysis
