1. DNA Amplification and PCR Technology
Prof. Dr Fridoon Jawad Ahmad
HEC Foreign Professor KEMU
Visiting Professor LUMMS-SSE

2. 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.

3. Gene Therapy

4. 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.

5. 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

6. The Discovery Werner Arber, Daniel Nathans and Hamilton Smith (1978 Nobel Prize)

7. Restriction Sites
Palindromic Sequences: A palindromic sequence is the same when read in 5′ to 3′ direction on either complementary strand of DNA.
DENNIS SINNED
GTAATG VS GTATAC

8. Blunt Ends Sticky Ends

9. Ligase/ Endonuclease

10. The Steps In Genetic Engineering

11. Many Proteins Collaborate at The Replication fork

12. The Mechanisms of DNA Replication

13. Requirements of DNA Replication
Template (Single stranded DNA)
Enzymatic Activity (DNA Polymerase)
Initiator (Primer/3’ hydroxyl group)
Spare parts (Nucleotide tri-phosphates)
Fourteen DNA Polymerases have been identified in humans and three in bacteria

14. PCR Technique and Applications

15. 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.

16. 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

17. Animation

18. Now & Then

19. 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.

20. The temperature profile of a PCR cycle is controlled by the thermal cycler program

21. Exponential Amplification
Cycles
Copies 1 2 4 16 10
1,024 15
32,768 20
1,048,576 25
33,554,432 30
1,073,741,824

22. Detection of PCR Product

23. Animation

24. Applications of PCR technology
Disease Detection
                      
Many pathogens can be detected from a small sample e.g.
HCV
HBV
HIV
Adenoviruses
Influenza virus
Cytomegalovirus etc
Isolation and detection of HCV RNA from whole blood

25. DNA Finger Printing in
Paternity

26. DNA Finger Printing in Crime Investigation

27. Real Time PCR (RT-PCR) primer
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.
The 3' phosphate group (P) prevents extension of the TaqMan probe.
The presence of the enzyme,Taq polymerase, enables extension of the primer which displaces the TaqMan probe.
The displaced probe is cleaved by Taq DNA polymerase resulting in an increase in relative fluorescence of the reporter.
Polymerisation is now complete.

28. Real Time PCR (RT-PCR)

29. DNA
Sequencing

30. Customized Medicine