NAME : MARIYA SHABBIR BARWANIWALA.

ROLL NO: 26

SY-BSC BIOTECHNOLOGY

SEMESTER-4

PAPER- 402.
DNA cloning is a laboratory technique that allows multiple copies of a
specific DNA segment to be created. In vitro site-specific
recombination is one method for cloning DNA. This method makes
use of specific recombinases' ability to recognise specific DNA
sequences and perform site-specific recombination between them,
such as the recombinase.

In vitro site-specific recombination begins with the creation of two

DNA fragments, each containing one half of the desired
recombination site. These fragments are then purified and mixed in
vitro, allowing the site-specific recombination reaction to take place.
The recombination reaction produces a single, continuous DNA
molecule containing the desired DNA segment.
When the recombination reaction is finished, the cloned DNA can be
introduced into the cell.

Numerous genome sequencing projects have identified a large

number of candidate open reading frames, many of which have no
known function. The transfer of DNA segments into a variety of vector
backgrounds for protein expression and functional analysis is typical
in the analysis of these genes. We describe recombinational cloning,
which uses in vitro site-specific recombination to achieve high-
efficiency directional cloning of PCR products and subsequent
automatic subcloning of the DNA segment into new vector backbones.
Numerous DNA segments can be transferred in parallel into a variety
of vector backgrounds, allowing for high-throughput, in-depth
functional analysis of genes and rapid protein expression
optimization. The resulting subclones retain orientation and reading
frame register, allowing amino-acid substitution.

DNA cloning using vitro site methods is a laboratory technique used to

produce multiple copies of a specific DNA sequence. There are several
methods for DNA cloning, including restriction digestion and ligation,
PCR (polymerase chain reaction) amplification, and recombinant DNA
technology.

Restriction Digestion and Ligation: This method involves cutting the

DNA of interest with specific restriction enzymes, which produce
specific recognition sites in the DNA. The cut DNA is then mixed with a
cloning vector, such as a plasmid, and ligated using a DNA ligase. The
ligated product is then transformed into bacteria, which serves as a
host for the cloning vector. The bacteria replicates the DNA,
producing multiple copies of the original DNA sequence.

PCR Amplification: This method uses a DNA polymerase and a heat-

stable primer to amplify a specific DNA sequence. The DNA is first
heated to denature it, allowing the primers to anneal to the target
sequence. The DNA polymerase then synthesizes new DNA strands,
resulting in the amplification of the target sequence. The amplified
DNA can then be cloned into a plasmid or another cloning vector.

Recombinant DNA Technology: This method involves introducing the

DNA of interest into a cloning vector, such as a plasmid, through
homologous recombination. The DNA of interest is first amplified by
PCR and the cloning vector is digested with the same restriction
enzymes used to cut the target DNA. The digested cloning vector is
then mixed with the amplified DNA, and the two are ligated. The
ligated product is then transformed into bacteria, which serves as a
host for the cloning vector. The bacteria replicates the DNA,
producing multiple copies of the original DNA sequence.

In vitro site methods have revolutionized the field of molecular

biology, allowing for the efficient and precise manipulation of DNA
sequences. These methods are widely used in research,
biotechnology, and medical applications, including the development
of new medicines and vaccines.
 REFERENCES

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC310948/