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Molecular Biology Sabah Linjawi ١
Lecture 11
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Molecular Biology Sabah Linjawi ٢
DNA cloning
DNA cloning is a technique to reproduce DNA fragments.
It can be achieved by two different approaches: (1) cell based, and (2) using polymerase chain
reaction (PCR).
In the cell-based approach, a vector is required to carry the DNA fragment of interest into the host cell.
The following figure shows a typical procedure by using plasmids as the cloning vector.
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Molecular Biology Sabah Linjawi ٣
Hosts and vectors
Most of the routine manipulations involved in gene cloning use Escherichia coli as the host organism
Plasmids and bacteriophages may be used as cloning vectors in E. coli
Vectors based on plasmids, viruses and
whole chromosomes have been used to carry foreign genes into other prokaryotic and
eukaryotic organisms
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Molecular Biology Sabah Linjawi ٤
The essential steps in DNA cloning using plasmids as vectors
a) DNA recombination. The DNA fragment to be cloned is inserted into a vector.
The recombinant vector must also contain an antibiotic-resistance gene.
(b) Transformation. The recombinant DNA enters into the host cell and proliferates.
It is called "transformation" because the function of the host cell may be altered.
Normal E. colicells are difficult to take up plasmid DNA from the medium.
If they are treated with CaCl2, the
transformation efficiency can be significantly enhanced. Even so, only one cell in about 10,000 cells may take up a plasmid DNA molecule.
(c) Selective amplification. A specific antibiotic is added to kill E. coliwithout any protection.
The transformed E. coli is protected by the antibiotic-resistance genewhose product can inactivate the specific antibiotic.
In this figure, the numbers of vectors in each E.
coli cell are not the same, because they may also reproduce independently.
(d) Isolation of desired DNA clones.
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Molecular Biology Sabah Linjawi ٥
Cloning Vectors
Vector" is an agent that can carry a DNA fragment into a host cell.
If it is used for reproducing the DNA fragment, it is called a "cloning vector".
If it is used for expressing certain gene in the DNA fragment, it is called an "expression
vector".
Commonly used vectors include plasmid,
Lambda phage, cosmid and yeast artificial
chromosome (YAC).
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Molecular Biology Sabah Linjawi ٦
Restriction enzymes
The procedure used in artificial DNA recombination is similar to the natural transpositional
recombination.
The major difference is that researchers can choose any appropriate enzymes to cut the DNA
molecules.
They are usually isolated from bacteria.
The role of these enzymes in bacteria is to "restrict"
the invasion of foreign DNA by cutting it into pieces.
Hence, these enzymes are known as restriction enzymes.
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Molecular Biology Sabah Linjawi ٧
Commonly used restriction enzymes
The "Recognition site" of a
restriction enzyme is also called the restriction site.
In this column, the first line is from 5' to 3' and the second line is from 3' to 5'.
The arrow indicates the cleavage site.
If the cleavage site is not at the center, the restriction enzyme will generate sticky ends, which can base-pair with other DNA fragments cleaved by the same restriction
enzyme.
If the cleavage site is at the center, the restriction enzyme will generate blunt ends.
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Molecular Biology Sabah Linjawi ٨
Agarose gel electrophoresis
Agarose gels separate linear DNA on the basis of size
By the migration of DNA through a matrix under the influence of an electric field
Electrophoresis may be used to determine the gross organization of plasmid molecules
Specific DNA fragments may be cut of agarose
gels and purified for use in subsequent cloning
experiments
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Molecular Biology Sabah Linjawi ٩
DNA Ligation
Joining linear DNA fragments together with covalent bonds is called ligation.
More specifically, DNA ligation involves creating a phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another.
The enzyme used to ligate DNA fragments is T4 DNA ligase, which originates from the T4
bacteriophage.
This enzyme will ligate DNA fragments having overhanging, cohesive ends that are annealed together
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Molecular Biology Sabah Linjawi ١٠
The EcoRI example
The EcoRI example below - this is equivalent to
repairing "nicks" in duplex DNA. T4 DNA ligase will also ligate fragments with blunt ends, although
higher concentrations of the enzyme are usually recommended for this purpose
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Molecular Biology Sabah Linjawi ١١
Transformation
Transformation is the process of take-up of foreign DNA, normally plasmids by bacteria
Plasmids are cloned by transfer into strains of E.coli with defined genetic properties
The E.coli cells can be made competent to take up plasmid DNA by treatment with Ca
2+
The cells are plated out on agar and grown to
yield single colonies or clones
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Molecular Biology Sabah Linjawi ١٢
Transformation
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Molecular Biology Sabah Linjawi ١٣
References
Molecular Biology. P.C. Turner, A.G. Mclennan, A.D.
Bates & M.R.H. White.School of Biological
Sciences, University of Liverpool, Liverpool, UK.
Second edition. BIOS Scientific Publishers, 2000.
www.vivo.colostate.edu/hbooks/genetics/.../ligation.
html
en.wikipedia.org/wiki/Transformation_(genetics
www.hawaii.edu/microbiology/MO/ligation.htm
