CHAPTER 4

AMPLIFICATION
OF DNA and DNA
analysis

At the end of the lecture, students

should be able to:
PART (a)
Describe the principles of PCR
Explain the steps in PCR process
Elaborate the applications of PCR
Understand RT-PCR (reverse
transcriptase vs Real time PCR)

What Is the Polymerase Chain

Reaction (PCR)?
What if you don't have enough DNA?

small sample
of DNA
serves as
template
for DNA
polymerase

The

Polymerase chain reaction (PCR)

Oligonucleotides complementary to a given
DNA sequence prime the synthesis of only
that sequence (primer)
Heat-stable Taq DNA polymerase survives
many cycles of heating.
Theoretically, the amount of the specific
primed sequence is doubled in each cycle.

Polymerase Chain Reaction

history

Polymerase chain reaction (PCR) is used to

amplify DNA and can be used to yield a DNA
fragment for cloning
Invented by Kary Mullis and colleagues in
1980s
Special heat-stable polymerases are now
used that are able to work after high
temperatures
Researchers no longer need to add fresh
DNA polymerase after each round of
replication as before

Standard PCR-Taq polymerase

Use enzyme DNA polymerase to copy a selected

region of DNA
The selected region of DNA now doubles in amount
with each synthesis cycle
Simplest protocol employs a heat-stable DNA
polymerase (Taq polymerase), originally isolated from
Thermus aquaticus, a bacterium that lives in hot
springs at temperatures >90 (higher than normal)

The steps involve:

Mix DNA sample with 4
deoxyribonucleotides (dNTP - dATP,
dTTP, dCTP, dGTP)
Taq polymerase
Also add a large excess of 2 short
synthetic DNA fragments
(oligonucleotides) complementary to DNA
sequences at the 3' ends of the DNA region
to be amplified
These short oligonucleotides serve as
primers to which nucleotides are added
during the following replication steps

More steps
Then heat the mixture to ~93C, melting
(denaturing) the DNA in mix & separating
the strands DENATURING STEP
The mix is then cooled to ~60C > allows
primers to bind 3' ends of both target DNA
strands ANNEALING STEP
Raise the temperature to ~72C > allows
the thermophilic polymerase to add
complementary nucleotides to the 3' end of
the primers ELONGATION STEP
As the polymerase extends the primers,
it selectively copies the target DNA to form
new complementary DNA strands

The concept..
Raised temperature causing newly formed
& original strands to separate from each
other
The sample is then cooled to allow
synthetic primers in mixture to bind
once again to the target DNA, which is now
present at twice the original amount
Repeat cycle over & over again, doubling
the amount of the specific DNA region
flanked by the bound primers each cycle

Finally
Generates large DNA amounts of this
specific DNA region from minute amounts
(billions of copies) in just a few hours
Uses a thermal cycler that automatically
changes the temperature of the reaction
mixture, allowing each step in the cycle to
take place
PCR can generate large amounts of DNA
from miniscule starting samples

Trouble shooting
If the bands obtained are numerous
(unspecific annealing), raise the annealing
temperature.
If no bands are obtained (too stringent),
lower the annealing temperature.
A working annealing temperature should be
around 3-5oC decrease from the lower Tm
value of the primer pairs.

The application of PCR

PCR has been used in criminal investigations
to generate DNA quantities from a spot of
dried blood left on crime suspect's clothing or
from DNA present as part of single hair follicle
left at crime scene
For this purpose, one selects regions of
genome for amplification that are highly
polymorphic
(i. e., vary at high frequency within the
population)
Same procedure can be used to study DNA
fragments from well-preserved fossil remains
that may be millions of years old

Using Reverse Transcriptase PCR

(RT-PCR) in cDNA Cloning
Cloning a cDNA from just one mRNA
whose sequence is known, is called
reverse transcriptase PCR (RT-PCR)
Difference between PCR and RT-PCR

Starts with an mRNA, not dsDNA

Begin by converting mRNA to DNA
Use reverse primer to produce 1st strand DNA
Use forward primers to convert ssDNA to dsDNA
Continue with standard PCR

Adding oligo dT adaptor/anchor

Reverse Transcriptase PCR (RTPCR) in cDNA Cloning

RT-PCR is used to clone expressed genes
byreverse transcribingthe RNA of interest.
Therefore, it is a method that helps to
quantify gene expression.
Can also be used to insert eukaryotic genes
into prokaryotes. Eukaryotic cells contained
introns, but not the mature mRNA.
Therefore, the product can be used for
protein production and purification.

RT-PCR to clone a
single cDNA
With care,
restriction enzyme
sites can even be
added to the ends of
the cDNA of interest
Able to generate
sticky ends for
ligation into vector
of choice
2 sticky ends permits
directional cloning

Real-Time PCR
Real-time PCR quantifies the
amplification of the DNA as it occurs
As the DNA strands separate, they anneal
to forward and reverse primers, PLUS a
fluorescent-tagged oligonucleotide
complementary to a part of one DNA
strand that serves as a reporter probe

Real-Time PCR

A fluorescenttagged
oligonucleotide
serves as a
reporter probe
Fluorescent tag
at 5-end
Fluorescence
quenching tag
at 3-end

Real-Time PCR

As PCR progresses from

the forward primer, the
5 tag is separated from
the 3 tag and allows the
5 tag to fluoresce

Fluorescence increases
with incorporation into
DNA product and can be
quantitated

Real-Time PCR
Increase of fluorescence
intensity is quantitated
by the Real time PCR
machine

DNA Separation Gel Electrophoresis

& Pulse Field Gel
Electrophoresis

At the end of the lecture, students

should be able to:

the importance of gel

electrophoresis
Differentiate three types of gel
electrophoresis
Understand the principles of gel
electrophoresis
Explain

Gel Electrophoresis

Gel electrophoresis provides a

powerful method to separate,
analyze and sometimes purify
proteins and nucleic acids.
Separations are based on sizes,
charges and conformation.

Gel Electrophoresis
Two main types of gels are agarose and
polyacrylamide
Polyacrylamide forms a tighter matrix and
is typically used to separate proteins;
smaller biomolecules such as short
sequences of DNA and RNA.
Agarose forms a looser, more open matrix
that enables separation of DNA molecules
from around 200-50,000 bp

Agarose gel electrophoresis

Agarose gels are prepared in a box equipped

with a removable comb.
Once agarose has gelled, comb will be
removed to expose slots/wells for DNA to be
placed or pipetted in together with dyes.
Ladders are placed for size referencing.
Agarose is placed inside the electrophoresis
apparatus and filled with a running buffer
(placed in horizontal position)
Electricity moved the DNA (negatively
charged) to the positive pole (anode).

Agarose gel electrophoresis

Agarose gel electrophoresis

DNA were separated based on difference in

sizes.

The principle of movement is friction.

Small DNA molecules encountered less
frictional drag therefore moving faster.
Larger DNA molecules had lower mobility
therefore will be at higher position.
DNA is stained with a fluorescent dye (often
EtBr) and visualized under UV
illumination.

Agarose gel electrophoresis

Polyacrylamide Gel Electrophoresis

(PAGE)
A cross-linker polymer of acrylamide.
SDS-PAGE is usually used to separate
proteins in vertically positioned
apparatus.
SDS (Sodium dodecyl phosphate) will be
used denature protein subunits so that
they do not bind to one another

Coat negative polypeptide so they move to anode

Masks the natural charge of protein so they move
according to mass

Polyacrylamide Gel Electrophoresis

(PAGE)

The gel needs to be prepared between two glass

plates.
Oxygen will inhibit the polymerization process

Gel Electrophoresis
A typical setup consists of a gel slab
sandwiched between two glass plates, with
the ends enclosed in upper and lower
reservoirs of buffer
Samples to be run are loaded in wells at the
top of the gel, in conjunction with tracking
dye.
An electrical voltage is applied between the
upper and lower reservoirs, causing the
samples to migrate down through the gel.
Stained with Coomassie Blue.

Electrophoresis Apparatus

Polyacrylamide Gel Electrophoresis (PAGE)

Overview (Pulse Field Gel

Electrophoresis PFGE)

Pulsed Field - any electrophoresis process that

uses more than one electric field alternatingly
Pulsed Field Gel Electrophoresis (PFGE) is a
technique used to separate especially long
strands of DNA (up till Mb) by length in order
to tell differences among samples.
It operates by alternating electric fields to run
DNA through a flat gel matrix of agarose (see
diagram).
PFGE has traditionally been used for gene
mapping and medical epidemiology.

Overview (Pulse Field Gel

Electrophoresis PFGE)

Overview (Pulse Field Gel

Electrophoresis PFGE)

Pulse Field Gel Electrophoresis

PFGE

Voltage are periodically switched among three

directions
i) Runs in the central axis
ii) Runs at the angle of 60o on the left side
iii) Runs at the angle of 60o on the right side
Takes longer than normal electrophoresis
Larger sizes DNA (30-50 kb) will run as a single,
large diffuse band.
Various length of DNA can be separated with
alternating
field
direction
eg
yeast
chromosomes with 0.2 till 2.2 Mb.

PFGE Results

DNA
hybridisation &
fingerprinting

DNA hybridisation & fingerprinting

DNA hybridisation
Explain the process of DNA
hybridisation
Describe the types of DNA
hybridisation.
Elaborate the applications of DNA
hybridisation in DNA fingerprinting
Describe

DNA hybridisation
in identifying specific
DNA fragments
Definition The ability of one
stranded nucleic acid to bind
to another single strand of
complementary base sequence
Helps

DNA hybridisation Southern

Blotting
A

method developed by Edward Sothern

Used restriction enzyme (preferably 6
base cutters eg EcoR1, HindIII) to cut
genomic DNA.
Electrophoresis of the fragments.
Denature with an alkaline solution
Transfer to a membrane
(nitrocellulose/nylon).
Hybridize to a specific probe.

DNA hybridisation Southern

Blotting
Probes

are used to identify a

desired gene segment from
among the thousands of
irrelevant ones
Two types are widely used
Polynucleotides (also called
oligonucleotides)
Antibodies

Polynucleotide Probes
Researchers used the cloned homologous gene
from another organism
Consider there is enough sequence

similarity to permit hybridization

Need to lower stringency of hybridization
conditions to tolerate some mismatches
High temperature, high organic solvent

concentration and low salt concentration are

factors that caused the hybridization of a
probe and a DNA with minimal mismatches

Protein-based Polynucleotide Probes

If homologous DNA from another organism is
absent..
If amino acid sequence is known, deduce a set
of nucleotide sequences to code for these
amino acids
Construct these nucleotide sequences
chemically using the synthetic probes
Can use several synthetic probes
Genetic code is degenerate with most amino
acids having more than 1 nucleic acid triplet
Must construct several different nucleotide
sequences for most amino acids

DNA fingerprinting
DNA

typing devised by Alec Jeffreys

manipulated minisatellites
(Fragment that contained sequences
of bases (10-60 bases) that are
repeated many times (5-50 times).
Individuals differ in the basic
patterns.
Based on Southern blotting, he
detected the minisatellites.

DNA
fingerprinting

DNA fingerprinting
DNA

fingerprinting can be used to

identify criminals.
DNA obtained from a crime scene that
matches with the detained suspect
will nail the suspect (no two
individuals have same patterns..)
DNA fingerprints have extreme
sensitivity.

DNA fingerprinting
DNA

fingerprinting can also be used to

establish parentage.
Parents will have similar VNTR
patterns with their offspring and so is
between siblings.
eg Ghanaian boy with a British mother
wants to find his mother
(mitochondrial DNA is maternally
inherited).

Immunoblots (Western blotting)

proteins.
Used antibodies/antiserum
as probes.
Used a second antibody that
is labelled for detection.
Electrophoreses

DNA
sequencing

DNA sequencing
the types of DNA sequencing
Understand the principles of DNA
sequencing
Explain the process of DNA
sequencing
Elaborate the applications of DNA
sequencing
Know

Maxam-Gilbert base destruction

method
sequencing is the determination
of the precise sequence of
nucleotides in a sample of DNA
Bases of a DNA molecule are
selectively destroyed
Not used very much anymore because
reagents are highly toxic and very
dangerous
DNA

Maxam-Gilbert base destruction

method
Used purified DNA (need not to be cloned)
Radioactive labelling at 5 end (adding
gamma-32P ATP.
Chemical treatment (four different chemicals)
generates breaks at a small proportion of one
or two of the four nucleotide bases in each of
four reactions (G, A+G, C, C+T).

DNA sequencing
The

most popular method is called the

dideoxy method or Sanger method
(named after its inventor, Frederick
Sanger, who was awarded the 1980
Nobel prize in chemistry [his second]
for this achievement)

The Procedure
The

DNA to be sequenced is prepared as a

single strand (needs denaturing by heat)
This template DNA is supplied with
a mixture of all four normal (deoxy)
nucleotides

dATP
dGTP
dCTP
dTTP
A short primer sequence complimentary to the 3
end.

The Procedure
tubes of all four dideoxynucleotides,
(modified nucleotides) each present in limiting
quantities and each labeled with a "tag" that
fluoresces a different color / radioactively
labelled (OH group absent)

Different

ddATP
ddGTP
ddCTP
ddTTP

DNA

polymerase I

The Procedure
Because

all 4 normal nucleotides are present,

chain elongation proceeds normally until, by
chance, DNA polymerase inserts a dideoxy
nucleotide (shown as colored letters)
instead of the normal deoxynucleotide
If the ratio of normal nucleotide to the
dideoxy versions is high enough, some DNA
strands will succeed in adding several
hundred nucleotides before insertion of the
dideoxy version halts the process.

The Procedure
At

the end of the incubation period, the

fragments are separated by length from
longest to shortest.
The resolution is so good that a difference of
one nucleotide is enough to separate that
strand from the next shorter and next longer
strand.
Each of the four dideoxynucleotides
fluoresces a different color when illuminated
by a laser beam and an automatic scanner
provides a printout of the sequence.

Overview of Sanger dideoxy method

The

dideoxy chain termination procedure

developed by Frederick Sanger begins with a
primer of precisely defined length that has
a radioactive 32P-phosphate label at its 5'
end.
The primer is designed to hybridize at the
3'-end of the template strand whose
sequence is to be determined.

Overview of Sanger dideoxy

method
Synthesis

of a new strand of DNA is

initiated from the primer, and a chain
termination process (incorporation of a
2'-,3'-dideoxynucleotide, which leaves no 3'OH group for further chain growth) is used to
stop synthesis selectively at any one of the
four DNA nucleotides.
This generates a series of fragments whose
relative lengths can then be used to read the
nucleotide sequence.

Overview of Sanger dideoxy method

Gene to be sequenced is used as a template for
the synthesis of new DNA strands, each
randomly terminating due to the incorporation
of a chain terminating dideoxynucleotide in 4
different reaction tubes.
This produces a population of molecules, each
terminating at a different site.
Running the products in each tube on a gel
allows the determination of where each
chain terminating dideoxynucleotide was
incorporated.

Thank you