Lecture 3

DNA
TECHNOLOGIES
Asst. Prof. Dr. Abdullahi Umar Ibrahim, PhD.
▪ In 1986, Dr. Alec Jeffreys made genetic
fingerprinting available to the public. In
1986 was when DNA was first used in a
criminal investigation by Dr. Jeffreys. The
investigation used genetic fingerprinting
in a case of two rapes and murders that
had happened in 1983 and 1986.
▪ Then in 1987, DNA evidence was first
used in the united states on a Florida
rapist man, tommie lee Andrews. After
using DNA evidence in his case, he was
then sentenced to 22 years in prison for
the rapes that he had committed.
 Biology and Society: Crime Scene
Investigations: Murders in a Small Town
On November 22, 1983, a
15-year-old girl was raped
and murdered on a quiet
country lane. Three years
later, another 15-year-old
girl was raped and
murdered. These crimes
happened in a small town
called Leicestershire, which
is located in the united
kingdom.

Lynda Mann (left) and Dawn Ashworth, the 15-year-old victims

of rapist and murderer Colin Pitchfork

Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings

They collected fingerprints
and connected them with
semen stains collected from
where the raping and
murders were located.
DNA fingerprinting of DNA
samples from suspects and
the crime scene proved one
man guilty and another man
innocent

Colin Pitchfork
Golden State Killer

With thirteen confirmed murders, fifty rape

victims, over two hundred burglaries, and
countless years terrorizing the state of
California, the Visalia Ransacker / East Area
Rapist / Golden State Killer was one of the
most prolific serial offenders in history

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC72
19171/#:~:text=In%20the%20Golden%20State%20K
iller,crimes%20of%20a%20single%20individual.
▪ Recombinant DNA, or rdna, is DNA
that is formed by combining DNA from
different sources through a process
called genetic recombination. Often,
the sources are from different
organisms. Generally
speaking, DNA from different
organisms has the same chemical
general structure. For this reason, it is
possible to create DNA from different
sources by combining strands.
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Application of Rdna technology
▪ Recombinant DNA has numerous applications in science and medicine.
▪ One well-known use of recombinant DNA is in the production of insulin. Prior to the
advent of this technology, insulin largely came from animals. Insulin can now be
produced more efficiently by using organisms like E. Coli and yeast. By inserting
the gene for insulin from humans in these organisms, insulin can be produced.
Recombinant DNA technology is used in a number of applications including vaccines,
food products, pharmaceutical products, diagnostic testing, and genetically engineered
crops.
▪ A number of other pharmaceutical products, like antibiotics and human protein
replacements, are produced by similar methods.
▪ Food Products
▪ A number of food products are
produced using recombinant DNA
technology. One common example is
the chymosin enzyme,
an enzyme used in making cheese.
Traditionally, it is found in rennet
which is prepared from the stomachs
of calves, but producing chymosin
through genetic engineering is much
easier and faster (and does not
require the killing of young animals).
Today, a majority of the cheese
produced in the united states is made
with genetically modified chymosin.
▪ Diagnostic testing
▪ Recombinant DNA technology is also
used in the diagnostic testing field.
Genetic testing for a wide range of
conditions, like cystic fibrosis and
muscular dystrophy, have benefited
from the use of rdna technology.
▪ Crops
▪ Recombinant DNA technology
has been used to produce both
insect- and herbicide-resistant
crops. The most common
herbicide-resistant crops are
resistant to the application of
glyphosate, a common weed
killer. Such crop production is
not without issue as many
question the long term safety of
such genetically engineered
crops.
 Host cell grown in
culture to form a clone
of cells containing the
“cloned” gene of interest
Gene of
Protein expressed from
interest
gene of interest
Copies of gene Protein harvested

Basic research
Basic
and various Basic
research
applications research
on gene
on protein

Gene for pest Gene used to alter Protein dissolves Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants up toxic waste attack therapy stunted growth
 The Process of Genetic
Recombination
▪ In the 1970s, scientists found a class of
enzymes that severed DNA in
specific nucleotide combinations. These
enzymes are known as restriction
enzymes. That discovery allowed other
scientists to isolate DNA from different
sources and to create the first artificial
rdna molecule. Other discoveries
followed, and today a number of methods
for recombining DNA exist.
1. A specific gene (for example, a human gene) is identified and isolated.
2.This gene is inserted into a vector. A vector is the mechanism by which the genetic
material of the gene is carried into another cell. Plasmids are an example of a common
vector.
3.The vector is inserted into another organism. This can be achieved by a number of
different gene transfer methods like sonication, micro-injections, and
electroporation.
4.After the introduction of the vector, cells that have the recombinant vector are isolated,
selected, and cultured.
5. The gene is expressed so that the desired product can eventually be synthesized, usually
in large quantities.
• Bacterial restriction
enzymes cut DNA molecules
at specific DNA sequences
called restriction sites
• A restriction enzyme usually
makes many cuts, yielding
restriction fragments
• The most useful restriction
enzymes cut DNA in a
staggered way, producing
fragments with “sticky ends.”
Figure 20.3-3
Restriction site
5 3
GAATTC
DNA CTTAAG
3 5
1 Restriction enzyme
cuts sugar-phosphate
backbones.
3 5
5 3

5 Sticky 3
3 5
end
5
3

2 DNA fragment added 3

from another molecule 5
cut by same enzyme.
Base pairing occurs.

5 3 5 3 5 3
G AATT C G AATT C
C TTAA G C TTAA G
3 53 5 3 5
One possible combination
3 DNA ligase
seals strands
5 3

3 Recombinant DNA molecule 5

Common Restriction Enzymes
Gene Cloning and Vectors
▪ Cloning: DNA or gene cloning is a process of duplicating of many copies of an exert copy of gene
or DNA using bacteria as a vector

▪ Gene cloning involves using bacteria to make multiple copies of a gene

▪ Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial
cell

▪ Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA

▪ This results in the production of multiple copies of a single gene

 Gene Cloning

GOAL: To get enough copies of the gene to manipulate

Gene Cloning vector Recombinant DNA

Host Multiply

Started with: few copies Ended with: Many copies.

All identical to starting gene - CLONES

 What Vectors
Have to Do With
Genes and
Cloning
▪ In molecular cloning, the vector is a
DNA molecule that serves as the
carrier for the transfer or insertion of
foreign gene(s) into another cell,
where it can be replicated and/or
expressed. Vectors are among
the essential tools for gene cloning and
are most useful if they also encode
some kind of marker gene encoding
a bioindicator molecule that can be
measured in a biological assessment to
ensure their insertion, and expression,
in the host organism.
▪ Specifically, a cloning vector is DNA taken
from a virus, plasmid, or cells (of higher
organisms) to be inserted with a foreign DNA
fragment for cloning purposes. Since the
cloning vector can be stably maintained in an
organism, the vector also contains features
that allow for the convenient insertion or
removal of DNA. After being cloned into a
cloning vector, the DNA fragment can be
further sub-cloned into another vector that
can be used with even more specificity.
▪ Plasmids, which are circular pieces of
DNA, are the most commonly used vectors
used to introduce foreign DNA into bacterial
cells. They often carry antibiotic resistance
genes that can be used to test for expression of
the plasmid DNA, on antibiotic petri plates.
The Major Types of Cloning Vectors
▪ Plasmid: Circular extrachromosomal DNA that autonomously replicates inside the
bacterial cell. Plasmids generally have a high copy number, such as puc19 which has a
copy number of 500-700 copies per cell.
▪ Phage: Linear DNA molecules derived from bacteriophage lambda. It can be replaced
with foreign DNA without disrupting its life cycle.
▪ Cosmids: Another circular extrachromosomal DNA molecule that combines features of
plasmids and phage.
▪ Bacterial Artificial Chromosomes: Based on bacterial mini-f plasmids.
▪ Yeast Artificial Chromosomes. This
is an artificial chromosome that contains
telomeres (disposable buffers at the ends
of chromosomes which are cut off during
cell division) with origins of replication,
a yeast centromere (part of a
chromosome that links sister chromatids
or a dyad), and a selectable marker for
identification in yeast cells.
▪ Human Artificial
Chromosome. This type of vector
is potentially useful for gene delivery
into human cells, and a tool for
expression studies and determining
human chromosome function. It can
carry a very large DNA fragment.
 plasmid
Plasmids are small circular DNA molecules that replicate separately from the bacterial
chromosome
 Structure
of vectors
▪ All engineered vectors have
an origin of replication (a
replicator), a cloning
site (located where the
insertion of foreign DNA
neither disrupts replication or
inactivation of essential
markers), and a selectable
marker (typically a gene that
provides resistance to an
antibiotic).
Gel Electrophoresis
▪ Can be used to separate the DNA
fragments obtained from different
sources.
 Gel electrophoresis system or
“gel box”

• DNA is loaded on the gel with electric current

• DNA has negative net charge and move from gel stained with
negative electrode to positive electrode ethidium bromide

• Smallest fragment will be farthest from the well

• Heavier fragment will be closer to the well
• Once DNA is separated, the gel is stained with
specific dye for viewing
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 The Polymerase Chain Reaction
(PCR)
▪ The polymerase chain reaction, PCR, can produce many copies of a specific
target segment of DNA
▪ A three-step cycle—heating, cooling, and replication—brings about a chain
reaction that produces an exponentially growing population of identical DNA
molecules
▪ The key to PCR is an unusual, heat-stable DNA polymerase called taq
polymerase.
Figure 12.14
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 DNA sequencing
▪ Up to 1990s, scientist sequence (reading
the sequence of DNA) using radiolabeled
nucleotide which are harmful
▪ Fred sanger developed sequencing method
used for human genome sequencing
project. These method is known as dideoxy
chain termination methods which is based
on the use of chain terminators known as
dideoxynucleotides (ddNTP)
▪ DdNTP differ from deoxynucleotides by the
lack of 3’ OH on the five carbon sugar
 Method
▪ Relatively short DNA fragments can be sequenced by the dideoxy chain
termination method, the first automated method to be employed
▪ Modified nucleotides called dideoxy ribonucleotides (ddNTP) attach to
synthesized DNA strands of different lengths
▪ Each type of ddNTP is tagged with a distinct fluorescent label that identifies
the nucleotide at the end of each DNA fragment
▪ The DNA sequence can be read from the resulting spectrogram
TECHNIQUE
DNA Primer Deoxyribonucleotides Dideoxyribonucleotides
(template strand) T 3 (fluorescently tagged)
G
5 C T dATP ddATP
T T
G 5 dCTP ddCTP
A
C DNA dTTP ddTTP
T polymerase
T dGTP ddGTP
C
G
A P P P P P P
C G G
A
A OH H
3
DNA (template Labeled strands
5 C strand) ddG 3
T ddA A
G ddC C C
A ddT T T T
C ddG G G G G
T ddA A A A A A
T ddA A A A A A A
C ddG G G G G G G G
G ddC C C C C C C C C
A T T T T T T T T T
C G G G G G G G G G
A T T T T T T T T T
3 A T T T T T T T T T 5
Shortest Longest
Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand
RESULTS
Last nucleotide G
of longest A
C
labeled strand T
G
A
Last nucleotide A
of shortest G
labeled strand C
TECHNIQUE
DNA Primer Deoxyribonucleotides Dideoxyribonucleotides
(template strand) T 3 (fluorescently tagged)
G
5 C T dATP ddATP
T T
G 5 dCTP ddCTP
A
C DNA dTTP ddTTP
T polymerase
T dGTP ddGTP
C
G
A P P P P P P
C G G
A
A OH H
3
TECHNIQUE (continued)
DNA (template Labeled strands 3
5 C strand) ddG
T ddA A
G ddC C C
A ddT T T T
C ddG G G G G
T ddA A A A A A
T ddA A A A A A A
C ddG G G G G G G G
G ddC C C C C C C C C
A T T T T T T T T T
C G G G G G G G G G
A T T T T T T T T T
3 A T T T T T T T T T 5
Shortest Longest
Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand
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 Study Question
▪ EXPLAIN HOW FINGERPRINT
CAN HELP SOLVE MURDER
▪ What is recombinant DNA
TECHNOLOGY?
▪ -What is a plasmid?
▪ Explain steps involve in pcr
▪ EXPLAIN THE MECHANISM OF
SANGER SEQUENCING
 Quiz
▪ https://byjus.com/biology/tools-recombinant-dna-technology/
 Assignment
▪ With the help of illustration, discusses about western, southern and northern
blotting.
▪ A case. Was brought to a judge regarding a paternity test. Mr. A claim that the
child doesn’t resembled him and he doesn’t feel chemistry between them. Thus,
he requested for paternity test. With the help of illustration discuss the following
results:
a) The child belong to him
b) The child doesn’t belong to him
▪ Assignment should be submitted next week. Use different colors to show
variations.