Restriction enzymes are classified as endonucleases.
Their biochemical activity is the hydrolysis ("digestion") of the
phosphodiester backbone at specific sites in a DNA sequence. By
"specific" we mean that an enzyme will only digest a DNA molecule
after locating a particular sequence.
Saturday, 29 September 2012
1. Better Crops (drought & heat resistance)
2. Recombinant Vaccines (ie. Hepatitis B)
3. Prevention and cure of sickle cell anemia
4. Prevention and cure of cystic fibrosis
5. Production of clotting factors
6. Production of insulin
7. Production of recombinant pharmaceuticals
8. Plants that produce their own insecticides
9. Germ line and somatic gene therapy
There are three different methods by which Recombinant DNA
is made.
1. Transformation
The first step in transformation is to select a piece
of DNA to be inserted into a vector. The second step is to cut that piece of
DNA with a restriction enzyme and then ligate the DNA insert into the vector
with DNA Ligase. The insert contains a selectable marker which allows for
identification of recombinant molecules. An antibiotic marker is often used so
a host cell without a vector dies when exposed to a certain antibiotic, and the
host with the vector will live because it is resistant.
The vector is inserted into a host cell, in a process
called transformation. One example of a possible host cell is E. Coli. The host
cells must be specially prepared to take up the foreign DNA.
Selectable markers can be for antibiotic resistance,
color changes, or any other characteristic which can distinguish transformed
hosts from untransformed hosts.
2. Non-Bacterial
Transformation
This is a process very similar to Transformation, which was described
above. The only difference between the two is non-bacterial does not use
bacteria such as E. Coli for the host.
In microinjection, the DNA is injected directly into the nucleus of the
cell being transformed. In biolistics, the host cells are bombarded with high
velocity microprojectiles, such as particles of gold or tungsten that have been
coated with DNA.
3. Phage Introduction
Phage introduction is the process of transfection,
which is equivalent to transformation, except a phage is used instead of
bacteria. In vitro packagings of a vector is used. This uses lambda or MI3
phages to produce phage plaques which contain recombinants. The recombinants
that are created can be identified by differences in the recombinants and
non-recombinants using various selection methods.
1. Cut DNA at specific DNA sequences
§ Symmetrical series of four to eight bases
§ Sticky Sticky or blunt ends or blunt ends
§ Restriction fragments-specific for each DNA
2. Origin – bacteria
§ How do they protect their own DNA?
3. Combined by DNA ligase
Recombinant DNA (rDNA) is a form of artificial DNA
that is created by combining two or more sequences that would not normally
occur together.
In terms of genetic modification, it is created
through the introduction of relevant DNA into an existing organismal DNA, such
as the plasmids of bacteria, to code for or alter different traits for a
specific purpose, such as antibiotic resistance.
It differs from genetic recombination in that it does
not occur through natural processes within the cell, but is engineered.
The recombinant DNA technique was first proposed by
Peter Lobban, a graduate student, with A. Dale Kaiser at the Stanford University
Department of Biochemistry.
The technique was then realized by Lobban and Kaiser;
Jackson, Symons and Berg; and Stanley Norman Cohen, Chang, Herbert Boyer and
Helling, in 1972–74.
Fig. Shows how a Recombinant DNA molecule is form
Recombinant DNA technology is one of the recent advances in biotechnology, which was
developed by two scientists named Boyer
and Cohen in 1973.
A
series of procedures that are used to join together (recombine) DNA segments. A
recombinant DNA molecule is constructed from segments of two or more different
DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a
cell and replicate there, either on its own or after it has been integrated
into a chromosome
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