DNA Cloning and Analysis

Chapter 1 :
Why Gene Cloning and DNA Analysis are Important
 Introduction
 Since the beginning of the last century,
scientists have been interested in genes.
 First, they wanted to find out:
◦ what genes were made of
◦ how they worked
◦ how they were transmitted from generation
to generation with the seemingly mythic
ability to control both heredity and
variation.
 Introduction
 Genes were initially thought of in
functional terms as “hereditary units
responsible for the appearance of
particular biological characteristics,
such as seed colour”
 However, the physical properties of
genes were unclear.
The early development of genetics

 It was not until the 1940s that genes

were shown to be made of DNA, and
that a workable physical and functional
definition of the gene – a length of DNA
encoding a particular protein – was
achieved
The early development of genetics

 Next, scientists wanted to find ways

to study in more detail: the structure,
behavior, and activity of genes.
 This required the simultaneous
development of novel techniques for
DNA analysis and manipulation.
 The early development of genetics

 Thesedevelopments began in the

early 1970s with the first
experiments involving the creation
and manipulation of recombinant
DNA. Thus began the recombinant
DNA revolution.
 The advent of gene cloning
 Cloning was a significant breakthrough in
molecular biology because it became possible
to obtain homogeneous preparations of any
desired DNA molecule in amounts suitable for
laboratory-scale experiments.
Gene manipulation and gene cloning
 The definition of recombinant DNA is “any
artificially created DNA molecule which brings
together DNA sequences that are not usually
found together in nature”.
 Gene manipulation refers to “any of a variety of
sophisticated techniques for the creation of
recombinant DNA and, in many cases, its
subsequent introduction into living cells”
 The propagation of recombinant DNA inside a
particular host cell so that many copies of the
same sequence are produced is known as
cloning.
Gene manipulation and gene cloning

 However, the gene manipulation

techniques is based on the behavior of
genetic material in the living cells
Before we go further in the course, we
have to know:
 DNA is a chain of nucleotides
 The length of a piece of DNA is measured
by the number of nucleotides in this
piece (i.e. 100 nucleotide)
 all nucleotides are composed of sugar,
phosphate and nitrogen base; so the only
difference between nucleotides is the
nitrogen base they contain (A, C, G or T),
Prof. Mohamed A. Karam
 Therefore, the term “base” is frequently
used instead of the very long heavy term
“nucleotide”
 Consequently, the length of any piece of
double-stranded DNA is expressed as
number of “base-pair” it carry
 Example: DNA containing 100 nucleotides is
said to be : 100 base-pair (100 bp) long
 Each 1000 bp equals 1 kbp

Prof. Mohamed A. Karam

 For a piece of DNA to act as a gene, it
must be
1. a reservoir of information (holds the
instruction for expressing a trait) in the
form of base sequence
2. The gene information has to be passed
essentially unchanged from generation
to generation in a process known as
replication
3. A gene can accept occasional changes, or
mutations. This allows organisms to
evolve.
Prof. Mohamed A. Karam
Dr. Mohamed A. Karam
DNA Replication
 Replication of DNA requires:
1. DNA template with an origin of
replication
2. Primer (provides 3`-OH
3. DNA polymerase enzyme
4. Deoxynucleotide triphosphate (dNTPs)
 DNA Replication
 Replication of DNA requires:
1. DNA template with an origin of replication
2. Primer (provides free 3`-OH for DNA-
polymerase)
3. DNA polymerase enzyme
4. Deoxynucleotide triphosphate (dNTPs)
The new DNA strand grows in the direction 5`—3`
 Gene Expression
➢ Gene expression is the process by which gene
products are made.
➢ The information in the gene is decoded and
expressed as a product.
➢ Most genes hold the information for making vital
cellular molecules called proteins.
➢ The expression of these genes requires two steps,
transcription and translation.

Prof. Mohamed A. Karam

❑ During synthesis, RNA growth is always in the 5`-to-
3` direction; in other words, nucleotides are always
added at a 3` growing tip. Because complementary
nucleic acid strands are oppositely oriented, the fact
that RNA is synthesized 5` to 3` means that the
template strand must be oriented 3` to 5`

Prof. Mohamed A. Karam

Prof. Mohamed A. Karam
 What is gene cloning?
1. A fragment of DNA,
containing the gene to be
cloned, is inserted into a
circular DNA molecule
called a vector, to
produce a recombinant
DNA molecule.
2. The vector transports the
gene into a host cell,
which is usually a
bacterium, although
other types of living cell
can be used.
 What is gene cloning?

3. Within the host cell the vector

multiplies producing numerous
identical copies, not only of
itself but also of the gene that it
carries.
4. When the host cell divides,
copies of the recombinant DNA
molecule are passed to the
progeny and further vector
replication takes place.
5. After a large number of cell
divisions, a colony, or clone, of
identical host cells is produced.
The advent of the polymerase chain reaction

❑ During the 1980s, when the excitement engendered

by the gene cloning revolution was at its height, it
hardly seemed possible that another, equally novel
and equally revolutionary process was just around the
corner.
❑ Kary Mullis invented the polymerase chain reaction
(PCR) an exquisitely simple technique that acts as a
perfect complement to gene cloning.
❑ PCR has made easier many of the techniques that
were possible but difficult to carry out when gene
cloning was used on its own.
 What is PCR?

❑ The polymerase chain reaction is very different from

gene cloning.
❑ Rather than a series of manipulations involving
living cells, PCR is carried out in a single test tube
simply by mixing DNA with a set of reagents and
placing the tube in a thermal cycler, a piece of
equipment that enables the mixture to be incubated
at a series of temperatures that are varied in a
preprogrammed manner.
❑ The basic steps in a PCR experiment are as follows
What is PCR?
Why gene cloning and PCR are so important

Obtaining a pure sample

of a gene by cloning
Why gene cloning and PCR are so important

PCR can also be used to purify a gene

 Why gene cloning and PCR are so important

❑ The methods led to rapid and efficient DNA

sequencing techniques that enabled the structures of
individual genes to be determined
❑ The methods led to procedures for studying the
regulation of individual genes, which have allowed
molecular biologists to understand how aberrations
in gene activity can result in human diseases such as
cancer.
❑ The techniques spawned modern biotechnology,
which puts genes to work in production of proteins
and other compounds needed in medicine and
industrial processes.
 Why gene cloning and PCR are so important

❑ It has extended the range of DNA analysis and enabled

molecular biology to find new applications in areas of
endeavor outside of its traditional range of medicine,
agriculture, and biotechnology.
❑ Archaeogenetics, molecular ecology, and DNA forensics
are just three of the new disciplines that have become
possible as a direct consequence of the invention of
PCR, enabling molecular biologists to ask questions
about evolution and the impact of environmental
change on the biosphere, and to bring their powerful
tools to bear in the fight against crime.