explain about the construction of recombinant DNA? need the answer very urgentlyy!!
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There are three different methods by which Recombinant DNA is made. They are
Transformation, Phage Introduction, and Non-Bacterial Transformation. Each
are described separately below.
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.
Different vectors have different properties to make them suitable to different
applications. Some properties can include symmetrical cloning sites, size, and
high copy number.
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.
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.
Transformation, Phage Introduction, and Non-Bacterial Transformation. Each
are described separately below.
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.
Different vectors have different properties to make them suitable to different
applications. Some properties can include symmetrical cloning sites, size, and
high copy number.
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.
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.
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