CHAPTER 4

Techniques in DNA
Technology
Syllabus Content
4.1 Introduction to DNA technology
4.2 DNA extraction
4.3 Gel electrophoresis
4.4 Polymerase Chain Reaction (PCR)
4.5 Recombinant DNA and DNA Cloning
4.5.1 Plasmid vector
4.5.2 Restriction endonucleases
4.5.3 DNA ligase
4.6 DNA Sequencing
4.6.1 Sanger method
4.7 Application of DNA technology
Introduction
DNA technology has launched a revolution in the area of biotechnology (the manipulation
of organisms or their components to make useful products).

Practices that go back centuries are forms of biotechnology: for example,

the use of microbes to make wine (from juice, using Yeast) and cheese (from milk, using Bacteria) and
the selective breeding of livestock, which exploits naturally occurring mutations and genetic recombination.

Modern biotechnology based on the manipulation of DNA in vitro differs from earlier
practices by enabling scientists to modify specific genes and move them between organisms
as distinct as bacteria, plants and animals.
What Is Biotechnology?
Harnessing the natural biological processes
of living systems for the benefit of mankind

• Biotechnology in the past

making bread (from flour, using Yeast) and cheese, brewing
beer (from grain, using Bacteria)
crossbreeding plants

• Modern biotechnology
– genetic engineering
 Genetic Engineering
or
Recombinant DNA Technology
 Genetic Engineering

The process of manipulating and transferring

instructions carried by genes from one cell to
another

Why do scientists want to change gene

instructions?
• to produce needed chemicals
• to carry out useful processes
• to give an organism desired characteristics
 DNA IS
EVERYWHERE
 DNA and Genes
A gene = is a segment of DNA that contains the
messages (instructions) to encode the
information necessary for producing
specific protein
Genes = the coding system for instructions

Guanine Cytosine
(G) (C)

Adenine Thymine
(A) (T)

bases
 Genes and Proteins

transcription

mRNA protein trait

translation
Gene
(a piece of DNA)
General Steps in Genetic Engineering
Sample preparation

Gel Electrophoresis

PCR

Recombinant DNA &

DNA Cloning

Sequencing
 1. Sample Preparation
Sample:
Can select the sample from various kinds of
plants, animals, microorganisms, tissue, etc.
Depends on our project interest.

DNA extraction:
DNA extraction is the removal of deoxyribonucleic
acid (DNA) from the cells of the sample (e.g
plants or organisms) in which it normally resides.
Instrumentation used in
DNA Extraction
This bead beater is used in the
breaking apart or "lysing" of cells in
the early steps of extraction in
order to make the DNA accessible.
Glass beads are added to an
eppendorf tube containing a
sample of interest and the bead
beater vigorously vibrates the
solution causing the glass beads to
physically break apart the cells.

Other methods used for lysing cells

include
a french press,
a sonication device and
liquid nitrogen .
French Pressure Cell Press
Sonicator device
Liquid nitrogen
 A centrifuge such as this
can spin at up to 15,000 rpm
to facilitate separation of the
different phases of the
extraction. It is also used to
precipitate the DNA after the
salts are washed away with
ethanol and or isopropanol.

microtubes
 Basic DNA Extraction
1. Lysis the cells containing the DNA of interest by:

Physical methods:
Bead beating, French pressure cell press,
sonicating or manual grinding with liquid nitrogen
(mortar and pestle method)

Chemical methods:
Vortexing with phenol is effective for breaking
down proteinaceous cellular walls.

The addition of a detergent/ surfactant (SDS,

Sodium Dodecyl Sulfate) is necessary to
remove/dissolve the lipid components of the
cell membrane.

DNA associated proteins, as well as other cellular

proteins, may be degraded with the addition of a
protease (e.g.: lysozyme, proteinase K).
2. Precipitation/ removal of the protein
Addition of a salt (ammonium/ sodium/
potassium acetate) to precipitate the
protein
When the sample is vortexed with phenol-
chloroform and centrifuged the proteins will
remain in the organic phase (removal of
proteins and other contaminants by
extraction)
3. DNA of interest is precipitated by mixing with cold ethanol or isopropanol and
then spin it by using centrifuge. The DNA is insoluble in the alcohol and will come
out of solution, and the alcohol serves as a wash to remove the salt previously
added.

Pipette out Aqueous solution

into a new tube and mixed
with isopropanol

Precipitated DNA

4. Wash the resultant DNA pellet with cold alcohol again and
centrifuge for retrieval of the pellet.
5. After pouring the alcohol off the pellet and drying, the DNA can be
re-suspended in a buffer such as Tris or TE.
6. Presence of DNA can be confirmed by electrophoresing on an
agarose gel containing ethidium bromide, or another fluorescent
dye that reacts with the DNA, and checking under UV light.
 2. Gel Electrophoresis

Separation of DNA fragments

according to size,
based on movement through a gel medium
when an electric field is applied.
THE PRINCIPLES OF GEL
ELECTROPHORESIS

DNA is a negatively charged molecule. If put into an electric field, it

will move from the −ve to the +ve pole.

A gel serves as the matrix for the movement of the DNA molecule.
The gel is a semisolid material with certain size pores.

If DNA is cut into fragments of different sizes, during

electrophoresis the smaller ones are able to move faster through
the pores of the gel than the larger ones. This creates a pattern
where the fragments are arranged according to their sizes from the
−ve to the +ve pole.
 Electrophoresis A gel box is used to separate DNA in
tank/chamber/box an agarose gel with an electrical
charge. When the red and black leads
are plugged into a power supply the
DNA migrates through the gel toward
the positive charge due to the net
Power supply
negative charge of the molecule.

Different sized pieces of DNA move at

Gel comb Gel cast different rates, with the larger pieces
moving more slowly through the
porous medium, thereby creating a
size separation that can be
differentiated in a gel.
 DNA negatively charged
Negatively charged phosphate
backbone of the DNA
 Make up the agarose gel
which the DNA will be put into

Agarose powder

Gel comb
Gel comb
Gel cast
 Step 1
Agarose is added to buffer and
heated to 95ºC (to melt it). After
cooling, it is poured into a pre-
prepared gel cast

Gel comb

Step 2
a comb is inserted immediately to
create wells for loading the samples

Step 3
Leave the gel to solidify

Step 4
the comb is removed slowly (see set
of wells that have been created)
Buffer solution added to the tank
Loading dye added to the DNA
DNA samples loaded into wells
Electrical field (voltage) applied to
the Tank
Each sample, a mixture of DNA molecules,
is placed in a separate well near one end of
the thin slab of gel.

The gel is bathed in a buffer solution, and

has electrodes attached to each end.

When the current is turned on, the

negatively charged DNA molecules move
toward the positive electrode, with shorter
molecules moving faster than longer ones.

Bands are shown here in orange, but on an

actual gel, DNA bands are not visible until a
DNA-binding dye is added.

The shortest molecules, having traveled

farthest, end up in bands at the bottom of
the gel.
DNA is stained using
ethidium bromide (EtBr)

Staining of DNA on gel in EtBr solution

Destain with dH2O

 PCR
Polymerase Chain
Reaction
a tool to generate a large
amount of a gene of interest
 What is the Polymerase Chain Reaction?

It’s an in-vitro technique used for making

multiple copies of a particular segment of DNA

The segment may represent a small part of a

large and complex mixture of DNAs:
e.g. a specific gene of a large bacterial genome

PCR

Genome : 4,000,000 bp Multiple cycles of After 30 cycles (< 2 h), more than
Target sequence : 200 bp heating and cooling a billion copies of the target
sequence can be produced from a
single starting DNA molecule
 Denaturation Annealing Extension
94 °C, 30 s 60 °C, 30 s 72 °C, 30 s

• Taq DNA Polymerase enzyme

• Forward primer & Reverse primer

• dNTP mixes
Deoxyribonucleotide triphosphate
mixes: dATP, dTTP, dGTP, dCTP

• MgCl2

• PCR buffer

• DNA template
3. Polymerase Chain Reaction (PCR)

The purpose of a PCR (Polymerase Chain

Reaction) is to synthesize a huge number of
copies of a gene.

This amplification is required to have

sufficient DNA fragments for cloning or for
sequencing.

The PCR comprises of three major steps

(denaturation, annealing and extension),
repeated in up to 40 cycles.

This reaction is performed on an automated

cycler, called PCR machine, designed to
heat and cool the tubes with the reaction
mixture in programmed time intervals.
With PCR, any specific target segment within one or
many DNA molecules can be quickly amplified (copied
many times) in a test tube.

The crucial development that made PCR widely usable

was the discovery of an unusual thermo-stable DNA
polymerase, first isolated from prokaryotes living in hot
springs (thermophilic organisms; Thermus aquaticus),
that could withstand the heat at the start of each PCR
cycle.
PCR Technique
The starting materials for PCR are
double-stranded DNA containing the target nucleotide sequence
to be copied,
a heat-resistant DNA polymerase,
all four nucleotides, and
two short, single-stranded DNA molecules that serves as primers.

One primer is complementary to one strand at one

end of the target sequence; the second is
complementary to the other strand at the other end
of the sequence.
Result
During each PCR cycle, the target DNA sequenced
is double.

By the end of the 3rd cycle, one-fourth of the

molecules correspond exactly to the target
sequence, with both strands of the correct length.

After 20 or so cycles, the target sequence molecules

outnumber all others by a billionfold or more.
Denaturation:
Heat briefly to
separate DNA
strands

Annealing:
Cool to allow primers
to form hydrogen
bond with ends of
target sequence

Extension:
DNA polymerase adds
nucleotides to the 3’
end of each primer
Gel electrophoresis of PCR Products
 4. Recombinant DNA
& DNA Cloning
Common methods for cloning pieces of DNA used bacteria
(most often, Escherichia coli) and their plasmids ( as a
vector).
Firstly, a plasmid is isolated from a bacterial cell, and then the
foreign DNA is inserted into it.
This resulting plasmid as a recombinant DNA molecule,
combining DNA from 2 sources.
The plasmid is inserted again into a bacterial cell, producing a
recombinant bacterium, which reproduces to form a clone of
identical cells.
The dividing bacteria replicate the recombinant plasmid and
pass it on to their descendant, the foreign gene is cloned at the
same time.
Recombinant DNA
Recombinant DNA PCR Cut

technology product/
Gene
plasmid

Recombinant bacterium
 CUTTING AND JOINING DNA
For this technique, two types of enzymes are used:
Restriction endonucleases (RE) that act as scissors to cut
DNA at specific sites.

DNA ligase that joins two DNA molecules

RE recognize specific sequences within DNA molecules and

cut at the sugar-phosphate backbone.

DNA ligase can join DNA fragments that have complementary

sticky ends or blunt ends.
Restriction endonucleases

As
biological
scissors
 Functions of cloning
Two basic purposes:
to make many copies of a particular gene
to produce a protein product.

Researchers can isolate copies of a cloned gene from bacteria for use in
basic research or to endow an organism with a new metabolic capability
(e.g a pest resistance).

A protein with medical uses, such as human growth hormone, can be

harvested in large quantities from bacterial cultures carrying the cloned
gene for the protein.
 5. Sequencing
The ultimate goal in mapping a genome is to determine
the complete nucleotide sequence of each chromosome.

If a pure preparation of many copies of a DNA fragment

up to ~800 base pairs in length is available, the
sequence of the fragment can be determined by a
sequencing machine.

Sequencing techniques, often called the

dideoxyribonucleotide chain-termination method –
developed by British scientist, Frederick Sanger.
dideoxyribonucleotide

ribonucleotide Deoxyribonucleotide dideoxyribonucleotide

dATP ddATP
dTTP ddTTP
dGTP ddGTP
dCTP ddCTP

DNA : Deoxyribonucleic acid Chain-terminating inhibitors of DNA

polymerase, used in the Sanger
method for DNA sequencing
1.

2.

3.

4.
Technique in sequencing
1. The fragment of DNA to be sequenced is denatured into single
strands and incubated in a test tube with the necessary ingredients
for DNA synthesis :
A primer designed to base pair with the known 3’ end of the template strand,
DNA polymerase,
4 deoxyribonucleotides (dATP, dTTP, dGTP, dCTP), and
4 dideoxyribonucleotides (ddATP, ddTTP, ddGTP, ddCTP), each tagged with a specific
fluorescent molecule.

2. Synthesis of each new strand starts at the 3’ end of the primer and
continues until a dideoxyribonucleotide is inserted, at random,
instead of the normal equivalent deoxyribonucleotide. This
prevents further elongation of the strand. Eventually, a set of
labeled strands of various lengths is generated, with the colour of
the tag representing the last nucleotide in the sequence.
3. The labeled strands in the mixture are separated by passage
through a polyacrylamide gel in a capillary tube (Capillary
electrophoresis), with shorter strands moving through faster.
A fluorescent detector senses the colour of each fluorescent tag
as the strands come through. Strands differing by as little as one
nucleotide in length can be distinguished.

4. Results:
The colour of the fluorescent tag on each strand indicates the
identity of the nucleotide at its end. The results can be printed
out as a spectrogram, and the sequence, which is
complementary to the template strand, can then be read from
bottom to top.
Printout from a DNA sequencer showing the peaks
and valleys of a portion of a sequenced gene that
correspond to the color-coded bases adenine A,
thymine T, guanine G and cytosine C.
 Automated DNA Sequencer

Left: A DNA sequencer at California State University, San Bernardino. Right: Door
of sequencer is open to show a gel plate inside.
Applications of DNA technology
Medical applications – diagnosis of diseases,
human gene therapy
Pharmaceuticals products
Forensic evidence
Environmental cleanup
Agricultural applications – animal husbandry
and “Pharm” animals, genetic engineering in
plants.
 Human Gene Theraphy
Insert RNA version of normal
Gene therapy using a retroviral allele into retrovirus
vector.
A retrovirus that has been rendered
harmless is used as a vector in this
Let retrovirus infect bone marrow
procedure, which exploits the ability of a cells that have been removed
retrovirus to insert a DNA transcript of from the patient and cultured.
its RNA genome into the chromosomal
DNA of its host cell.

If the foreign gene carried by the Viral DNA carrying the normal
retroviral vector is expressed, the cell allele inserts into chromosome
and its descendants will possess the
gene product, and the patient may be
cured.

Cells that reproduce throughout life,

such as bone marrow cells, are ideal
candidates for gene therapy.
Inject engineered
cells into patient
Diagnosis of diseases

RFLPs (Restriction fragment

length polymorphism) as
markers for disease–causing
alleles.

This diagram depicts homologous

segments of DNA from a family in
which some members have a
genetic disease.

In this family, different versions of a

RFLP marker are found in
unaffected family members and in
those who exhibit the disease.

If a family member has inherited the

version of the RFLP marker with
two restriction sites near the gene
(rather than three), there is a high
probability that the individual has
also inherited the disease–causing
allele.
Forensic evidence
DNA fingerprints from a murder case.

This autoradiograph shows that DNA in

blood from the defendant′s clothes matches
the DNA fingerprint of the victim but differs
from the DNA fingerprint of the defendant.

This is evidence that the blood on the

defendant′s clothes came from the victim,
not the defendant.

The three DNA samples were subjected to

Southern blotting using radioactive probes.

The DNA bands resulting from

electrophoresis were exposed to probes
(after Southern blotting) for several
different RFLP markers in succession, with
the previous probe washed off before the
next one was applied.
Forensic evidence
Paternity Case

Schematic drawing showing DNA

fingerprints (profile) used in a paternity
dispute.

RFLP patterns (DNA fragments or

bands) of Father, Mother & two children
are shown here.

The profile shows that the Child A has

inherited one DNA band from each
parent but Child B has one band
inherited from the Mother and an
unrecognised band different from the
Father.

Child B is therefore not the child of the

Father.
‘Pharm’ animals
These transgenic sheep
carry a gene for a human
blood protein, which they
secrete in their milk.

This protein inhibits an

enzyme that contributes to
lung damage in patients
with cystic fibrosis and
some other chronic
respiratory diseases.

Easily purified from the

sheep′s milk, the protein is
currently under evaluation
as a treatment for cystic
fibrosis.
The goal of genetically
modifying animals is
usually that of
traditional breeding.

Better wool from sheep,

leaner meat from pigs,
fish that mature in
shorter amount of time.
 Genetic engineering in plants
Agrobacterium tumefaciens Plant cell
Genomic DNA
Genomic DNA (carries the gene of
interest)

Restriction
Ti plasmid Restriction
enzyme A
enzyme A

+
Empty Gene of
plasmid interest

Ti plasmid with the gene of interest

Ti plasmid with the new gene
cell’s
+ DNA Transformation

Agrobacterium
Plant cell

The new
gene

Cell division
Transgenic plant
THE END