DNA CLONING and APPLICATION
What is DNA cloning?
Cloning is the method of making precise duplicates of an organism or gene in biology. Producing an exact
replica of DNA is known as DNA cloning. In medical research, DNA cloning is frequently utilized to study
disease states and develop medications and treatments.
Cloning Process Overview
• DNA is extracted from the organism under study and cut into small fragments suitable for cloning.
• Restriction enzymes are used to cut the DNA at specific target sequences, creating recombinant DNA.
• The vector is cut with the same restriction enzyme used to cut the donor DNA.
• The complementary ends of both types of DNA unite randomly in a test tube.
• DNA ligase is used to seal the sugar-phosphate backbone of the DNA, forming a continuous and stable
double helix.
• The mixture contains a population of vectors each containing a different donor insert.
• Live bacterial cells are mixed with the mixture, and recombinant molecules enter living cells in a
process called transformation.
• The original mixture of transformed bacterial cells is spread out on the surface of a growth medium in
a flat dish (Petri dish).
• Each cell clone is a DNA clone because the recombinant vector has been amplified by replication
during every round of cell division.
• Each colony is a cell clone, but it is also a DNA clone because the recombinant vector has now been
amplified by replication during every round of cell division.
• A cDNA library is created by treating mRNA molecules with the enzyme reverse transcriptase, which is
used to make a DNA copy of an mRNA.
• Both genomic and cDNA libraries are made without regard to obtaining functional cloned donor
fragments.
• Several bacterial viruses have been used as vectors, with the lambda phage being the most commonly
used.
• Vectors are chosen depending on the total amount of DNA that must be included in a library.
Creating the clone
Cloning is a process that involves extracting DNA from an organism and cutting it into small fragments
suitable for cloning. Restriction enzymes are used to cut the DNA at specific target sequences, creating
recombinant DNA. The vector is then cut with the same restriction enzyme used to cut the donor DNA,
creating a space for the insertion of the donor DNA segment. The complementary ends of both types of
DNA unite randomly in a test tube, forming a stable double helix.
The mixture contains a population of vectors each containing a different donor insert. This solution is
mixed with live bacterial cells that have been specially treated to make their cells more permeable to
DNA. Recombinant molecules enter living cells through a process called transformation, where only a
single recombinant molecule enters any individual bacterial cell. Once inside, the recombinant DNA
molecule replicates like any other plasmid DNA molecule, and many copies are produced. When the
bacterial cell divides, all of the daughter cells receive the recombinant plasmid, which again replicates in
each daughter cell.
Another type of library is a cDNA library, created by treating messenger ribonucleic acid (mRNA) instead
of DNA. This mRNA molecules are treated with the enzyme reverse transcriptase, resulting in
complementary DNA (cDNA). A cDNA library represents a sampling of the transcribed genes, while a
genomic library includes untranscribed regions.
Both genomic and cDNA libraries are made without regard to obtaining functional cloned donor
fragments. Genomic clones do not necessarily contain full-length copies of genes, and genomic DNA
from eukaryotes contains introns, which are regions of DNA that are not translated into protein and
cannot be processed by bacterial cells. Expression libraries can be produced by slicing cDNA inserts
adjacent to a bacterial promoter region on the vector, allowing for several important technological
applications in DNA sequencing.
Several bacterial viruses have also been used as vectors, such as lambda phage, which can carry larger
inserts than either pUC plasmids or lambda phage alone. Vectors are chosen depending on the total
amount of DNA that must be included in a library.
Isolating the clone
Cloning is a process to obtain a clone of a specific gene or DNA sequence. The next step is to isolate the
clone among other members of the library, which may include the entire genome of an organism. A
cloned DNA segment that shows homology to the sought gene is often used as a probe. For example, a
mouse gene can be used to find an equivalent human clone from a human genomic library. Bacterial
colonies in a library are grown in Petri dishes, and a porous membrane is laid over the surface. DNA is
separated into single strands, and the probe is labeled with radioactive phosphorus. The probe DNA
adheres only to the clone containing the equivalent gene. The clone is then retrieved from the original
Petri dishes.
https://www.britannica.com/science/recombinant-DNA-technology
