St.

Louis University
Baguio City, Philippines
A.Y. 2014-2015

Application of Biotechnology on
Genetic Engineering (DNA Cloning)

Submitted by: Jerome T. Magno

Submitted to: Engr. Katelyn G. Gabon
DNA Cloning

DNA cloning is the starting point for many genetic engineering

approaches to biotechnology research.

Large amounts of DNA are needed for genetic engineering.

Multiple copies of a piece of DNA can be made either by using polymerase
chain reaction (PCR) or by cloning DNA in cells.

The polymerase chain reaction (PCR) is a technique that is

used to make multiple copies of a piece of DNA.PCR is carried out in
vitro, but mimics what happens in cells when DNA is copied (replicated)
prior to cell division.

How does PCR work?

Before PCR can occur, the two strands in the DNA double helix
need to be separated. This is called denaturation. It is done by raising
the temperature of the DNA solution. This causes the hydrogen bonds
between the complementary DNA chains to break, and the two strands
separate.

Next, the temperature is lowered and an enzyme (usually Taq

polymerase) joins free DNA nucleotides together. The order in which these
nucleotides are joined to the new strand is determined by the sequence
of nucleotides in the original DNA strand which is being copied.

The result is a double stranded DNA molecule which contains one newly
made strand and one original strand.

Next, the newly created double helix is separated (by heating the
solution) and the cycle is repeated.

How is DNA cloned in cells?

To get multiple copies of a gene or other piece of DNA you must isolate,
or cut, the DNA from its source and then paste it into a DNA vector
that can replicate (or copy) itself.

The four main steps in DNA cloning are:

1. The chosen piece of DNA is cut from the source organism using
restriction enzymes.

In the laboratory, restriction enzymes (or restriction endonucleases)

are used to cut DNA into smaller fragments. The cuts are always made at
specific nucleotide sequences. Different restriction enzymes recognize
and cut different DNA sequences.
Like all enzymes, a restriction enzyme works by shape-to-shape matching.
When it comes into contact with a DNA sequence with a shape that matches
a part of the enzyme, called the recognition site, it wraps around the
DNA and causes a break in both strands of the DNA molecule.

Each restriction enzyme recognizes a different and specific recognition

site, or DNA sequence. Recognition sites are usually only short - 4-8
nucleotides.

2. The piece of DNA is pasted into a vector and the ends of the
DNA are joined with the vector DNA by ligation.

In cells and in the lab, enzymes called ligases are used to join fragments
of DNA together. Only DNA fragments that have matching, complementary
ends can be joined by ligation.

Ligases join fragments of DNA together by catalyzing the formation of

bonds between neighboring nucleotides.

Ligation in cells

Cells naturally carry out ligation during DNA replication, when the
Okazaki fragments are joined together. Cells also use ligation to repair
DNA that has been damaged, either by normal cell metabolism or by
environmental factors, such as UV light or radiation. Up to 1 million
breaks can occur in the DNA of a single human cell each day.

3. The vector is introduced into a host cell, often a bacterium or

yeast, by a process called transformation. The host cells copy the
vector DNA along with their own DNA, creating multiple copies of
the inserted DNA.

Bacteria are commonly used as host cells for making copies of DNA in the
lab because they are easy to grow in large numbers. Their cellular
machinery naturally carries out DNA replication and protein synthesis.

4. The vector DNA is isolated (or separated) from the host cells DNA
and purified.

DNA that has been cut and pasted from an organism into a vector is
called recombinant DNA. Because of this, DNA cloning is also called
recombinant DNA technology.

What is cloned DNA used for?

DNA cloning is used to create a large number of copies of a gene or

other piece of DNA. The cloned DNA can be used to:
 Work out the function of the gene
Investigate a genes characteristics (size, expression, tissue
distribution)
Look at how mutations may affect a genes function
Make large concentrations of the protein coded for by the gene

Personal Insights

Biotechnology has help me to become knowledgeable of the

things and occurrences in my system and certain processes. One of these
is the nucleic acids, one of the biological molecules, which have two
types. One of this is the DNA (Deoxyribonucleic Acid) that we know is
present in all cells. In, genetic engineering in which it involves DNA
cloning, my understanding about this topic has broaden and we can call
it sharp because of my background of Biotechnology. In DNA cloning first
is we have to separate the two strands in the double helix structure of
DNA and then we imply an enzyme to join the free nucleotides together
in order of the original sequence and we learned that in the DNA
complementary base pairing the adenine (A) is paired to thymine (T),
Guanine (G) is then paired to Cytosine (C). So in the process of DNA
cloning the DNA is cut by using restriction enzymes and one example cited
in our lecture is the enzyme helicase that it separates the 2 DNA strands
by breaking the weak hydrogen bonds. And after it is introduced into a
vector and it will imply the process of ligation in which we imply the
use of an enzyme example is Ligase join fragments of DNA together. And
we know that in the cell there is a DNA replication, and in this ligation
carries out when the Okazaki fragments (series of short segments on the
lagging strand) are joined together. And so it will introduced to a host
cell or a vector that copy the vector DNA along with their own DNA,
creating multiple copies of the inserted DNA as well as the DNA
replication implies. And after the copying the DNA vector is separated
to the host cells just like the relationship of a substrate and an
enzyme, the substrate will go to its respective enzyme and when the
process is completed or one is benefited, the substrate will separate
into the enzyme and other reaction will the occur. And there is a
formation of cloned DNA or what we call the recombinant DNA. So, we then
employed biotechnology terms and applications in understanding the DNA
cloning. Biotechnology is crucial in our lives, it will open new
opportunities to understand and develop our world, and it will create
improvements and innovations to further make our life easier.