DNA Amplification and
PCR Technology
Prof. Dr Fridoon Jawad Ahmad
HEC Foreign Professor KEMU
Visiting Professor LUMMS-SSE
Uses of Amplified DNA
• SEQUENCING: Sequencing determines the base pair
sequence of a gene. By reading the 3-letter code,
sequencing also describes the AMINO ACID
SEQUENCE translated from that gene.
• MUTATION: It is possible to study the effects of
SINGLE AMINO ACID CHANGES on the function of
the gene product, which is, after all, the ultimate
purpose of the exercise (reverse genetics).
• MASS PRODUCTION OF PROTEINS: To use the
amplified gene to make HUMONGOUS QUANTITIES
of the GENE PRODUCT for commercial purposes.
• TRANSGENICS: INSERT the gene into ANOTHER
SPECIES.
• Gene Therapy
The DNA Analysis Dilemma
• Genomes are composed of large DNA chunks on the
order of millions of units.
• When scientists first considered studying genomes
they were faced with a problem: how to reproducibly
cut a genome’s DNA into fragments that were small
enough to handle?
• Scientist can only handle pieces of DNA a few
thousand units long
• Random cutting using chemical or mechanical
means was not a satisfactory way to obtain smaller
pieces of DNA.
• It was impossible to tell what the original order of the
DNA fragments were.
Restriction Endonucleases
• It was discovered that a type of bacterial enzyme was
found to have the ability to cut DNA in a test tube.
• These restriction endonucleases, cut double
stranded DNA at specific sites.
• In a bacterial cell, restriction endonucleases
(restriction enzymes) act as a kind of immune
system, protecting the cell from the invasion of
foreign DNA (virus).
• Many recombinant DNA technologies, which the field
of biotechnology heavily relies on, are unlikely to
have been developed without the discovery of
restriction enzymes
The Discovery
Werner Arber, Daniel Nathans and Hamilton Smith (1978 Nobel Prize)
Restriction Sites
• Palindromic Sequences: A palindromic sequence is
the same when read in 5′ to 3′ direction on either
complementary strand of DNA.
GTAATG
DENNIS SINNED
VS
GTATAC
Blunt Ends Sticky Ends
Ligase/ Endonuclease
The Steps In Genetic Engineering
Many Proteins Collaborate at The
Replication fork
The Mechanisms of DNA Replication
Requirements of DNA Replication
1. Template (Single stranded DNA)
2. Enzymatic Activity (DNA Polymerase)
3. Initiator (Primer/3’ hydroxyl group)
4. Spare parts (Nucleotide tri-phosphates)
Fourteen DNA Polymerases have been identified in humans
and three in bacteria
PCR Technique and
Applications
The Inventor
Kary Mullis
The idea for PCR is credited to Kary Mullis who was a
research scientist in the 1980s at a California biotechnology
company called Cetus (bought out by Chiron). Mullis, and five
other researchers in the Human Genetics Department at
Cetus, demonstrated that oligonucleotide primers could be
used to specifically amplify defined segments of genomic
DNA (or cDNA). Mullis was co-winner of 1993 Nobel Prize in
Chemistry.
Biochemistry of the Polymerase Chain Reaction
The logic of the PCR protocol follows directly from well-understood principles of
nucleic acid biochemistry. The basic components of a PCR reaction are:
- one or more molecules of target DNA
- two oligonucleotide primers
- thermostable DNA polymerase
- dNTPs
Animation
Now & Then
The PCR Amplification Cycle
Each PCR cycle requires three temperature steps to complete
one round of DNA synthesis. Before cycle 1 can be initiated, the
double-stranded DNA target needs to be heat-denatured to
provide single-stranded regions for primer annealing.
The temperature profile of a PCR cycle is
controlled by the thermal cycler program
Exponential Amplification
Cycles
1
2
4
10
15
20
25
30
Copies
2
4
16
1,024
32,768
1,048,576
33,554,432
1,073,741,824
Detection of PCR
Product
Animation
Applications of PCR technology
Disease Detection
Many pathogens can be
detected from a small sample
e.g.
Isolation and detection of HCV
RNA from whole blood
HCV
HBV
HIV
Adenoviruses
Influenza virus
Cytomegalovirus etc
DNA Finger
Printing
in
Paternity
DNA Finger Printing
in
Crime Investigation
Real Time PCR (RT-PCR)
primer
1. PCR primers 1 and 2 and a TaqMan probe,labelled with a reporter dye, (R)
and a quencher dye, (Q), bind to the DNA template.
2. The 3' phosphate group (P) prevents extension of the TaqMan probe.
3. The presence of the enzyme,Taq polymerase, enables extension of the
primer which displaces the TaqMan probe.
4. The displaced probe is cleaved by Taq DNA polymerase resulting in an
increase in relative fluorescence of the reporter.
5. Polymerisation is now complete.
Real Time PCR (RT-PCR)
DNA
Sequencing
Customized
Medicine