The document summarizes Kary Mullis' invention of polymerase chain reaction (PCR) which won him the 1993 Nobel Prize in Chemistry. PCR is a technique used to amplify a specific DNA sequence by using DNA polymerase to replicate that sequence many times. It involves repeated cycles of heating and cooling DNA to separate the double helix, followed by primer annealing and strand extension. This allows for exponential amplification of target DNA, making it useful for applications like DNA sequencing and cloning. The document also briefly introduces the CRISPR/Cas9 system, noting its use of a guide RNA and Cas9 nuclease to target and cleave specific DNA sequences.

1. LOGO
TRINH MAI DUY LUU
Class: Advanced Bioscience and Biotechnology Frontiers
Professor: Dr. Yuji SAITO
1

2. CONTENTS
The Nobel Prize in Chemistry 1993
Polymerase Chain Reaction (PCR)
Modified PCR techniquesModified PCR techniques
Conclusion
2

3. I
3

4. Dr. Kary Banks MullisI.1
Born: December 28, 1944 in Lenoir, North
Carolina
PhD. Biochemistry 1972, University of California
Founder and Chief Scientific Officer,
Altermune, LLC
The Nobel Pize in Chemistry 1993 and many
4
The Nobel Pize in Chemistry 1993 and many
other famous prizes
Scientific advisor for Health Discovery
Corporation-Georgia, Fondazione Villa Maria-Italy,
Palazzo Innovazione Project-Italy, Biological
Targets-Texas
Many Patents
Two book and many scientific papers
Kary B. Mullis

5. The Nobel Prize in Chemistry 1993I.2
The Nobel Prize in Chemistry 1993 was awarded "for contributions to the
developments of methods within DNA-based chemistry“ jointly with one half
5
developments of methods within DNA-based chemistry“ jointly with one half
to Kary B. Mullis "for his invention of the polymerase chain reaction (PCR)
method" and with one half to Michael Smith "for his fundamental
contributions to the establishment of oligonucleotide-based, site-directed
mutagenesis and its development for protein studies”
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/

6. II
6

7. DNA REPLICATION PROCESS
•Mechanisms of DNA replication in prokaryotes
and eukaryotes are essentially similar with some
differences due to differences in cell structure and
function between the two groups.
II.1
• DNA replication process includes initiation, DNA
synthesis and termination steps
7

8. DNA synthesis step – the replication fork
•Two DNA
polymerases, each
catalayzes the synthesis
of one daughter strand
• DNA primases
synthesize primers
• Single-stranded
II.1
• Single-stranded
Binding Proteins (SSB)
bind to the separated
parental strand
• DNA topoisomerase
unwinds the double
helix ahead the moving
replication fork
8
• The double helix must be separated into two
single strands by DNA helicases
Figure 1. The replication fork

9. Nucleic acid thermodynamics
• Hybridization,
interation between
complementary
strands of DNA into a
single complex
• Denaturation or
DNA melting, double-
II.2
DNA melting, double-
stranded DNA
unwinds and
separates into single-
stranded strand
• Annealing, binding
of a DNA probe or
primer to a DNA
strand
9
“Copyright 1997 from Encyclopedia of life sciences. Nucleic
acids: Thermal Stability and Denaturation”
Figure 2. “Melting curve” being monitored in a
absorbance experiment as a function of temprature.
Nucleic acids absorb strongly in the UV region and this
absorbance increases upon the disassociation of the
strands. Tm: the melting temprature

10. Polymerase Chain Reaction
II.3
A Polymerase Chain Reaction includes these following
elements:
•Primers are oligonucleotides (18-24 bps) which can
anneal with the target sequence specifically
•DNA template contains the target sequence
10
•DNA template contains the target sequence
•Enzyme DNA polymerase which now is purified from
the thermophilic bacterium Thermus aquaticus is named
Taq polymerase
•[Mg2+] is cation working as cofactor of Taq polymerase
•dNTPs include ATPs, GTPs, CTPs, and TTPs

11. Polymerase Chain Reaction
II.3
95oC
55oC
72oC
Figure 3. The basic PCR.
Duplex DNA is heat denatured
to give single strands, and two
oligonucleotide primers are
annealed to their
complementary sequences on
the target DNA. Taq
polymerase (thermosable) is
used to synthesis
11
“Copyright 2008 from An introduction to genetic engineering. Published by Cambridge”
used to synthesis
complementary strands from
the template strands by primer
extension. The cycle is then
repeated by denaturation of the
DNA, and the programmes is
repeated many times.

12. III
12

13. Application of PCR
III.1
13
“Copyright 2008 from An introduction to genetic engineering. Published by Cambridge”
Figure 4. Nuber of publications citing the polymerase
chain reaction from 1985 to 1999. This shows the rapid
spread of the use of PCR in research.

14. PCR TECHNIQUESIII.2
14

15. IV
15

16. Conclusion
The invention of Kary B. Mullis is extremely essential for
the development of science
There are many diverse applications of the PCR technique
IV.1
16

17. 17

18. CRISPR/CAS9
18

19. CRISPR/CAS9 system
Cas 9 is nuclease which is able to cleavage
dsDNA with two nuclease domains: RuvC-
like domian and HNH motif
sgRNA, chimeric sigle-guide RNA, includes
crRNA and tracrRNA
crRNA is able to bind directly to a 20-nucleotide
sequence, so called protospacer
tracrRNA, trans-activating CRISPR RNA), is able to
interact with crRNA and CAS9 protein
PAM (protospacer-adjacent motif) includes
3-nucleotide element with the sequence NGG
19

20. CRISPR/CAS9
Step 1
20
Cas9
nuclease
sgRNA
(single guide
RNA)
Cas9
complex

21. CRISPR/CAS9
Step 2
21

22. CRISPR/CAS9
Step 3
22

23. 23