PCR allows for the amplification of small amounts of DNA, generating millions of copies. It involves three steps - denaturation of the DNA template, annealing of primers, and extension of the primers by DNA polymerase. Repeating this process results in exponential growth in the number of DNA copies. DNA sequencing, such as the Sanger method, is used to determine the exact order of nucleotides in a DNA fragment and can be used to map genomes and detect genetic variations.

1. Lecture 31:
Molecular techniques
III. PCR and DNA sequencing
Readings (chapter 10)
Course 281

2. Lessons for life

3. AIMS
• Understand the process of PCR and the
components needed for the reaction.
• Understand why PCR is important in molecular
biology.
• Understand the importance of DNA
sequencing.
• Understand Sanger sequencing method as an
example of DNA sequencing.

4. Polymerase chain reaction
• Polymerase Chain Reaction
(PCR) allows the amplification
(copying) of small amounts of
DNA millions of copies.
• The method was developed
by Kary Mullis (1983) and he
was awarded the Nobel Prize
for his invention.

5. Polymerase chain reaction
• The process of PCR is
similar to the process of DNA
replication except it is done in
tubes rather than living cells.
• It is considered in many
cases the first step before any
genetic analysis.
• Many methods and
applications involve PCR.

6. DNA replication and PCR
• DNA replication in the cells involves making an
identical copy of the genome (DNA).
• PCR uses the same procedure but to generate
millions of copies of a small section of the
genome in a tube!

7. Why PCR?
PCR is used:
1. To amplify small quantities of DNA.
2. For DNA quantification.
3. For genetic profile analyses:
• RFLP
• Microsatellite
• Mitochondrial DNA genotyping
and sequencing.
4. For sequencing small section of the
genome or the genome.

8. Components
What do we need to replicate (copy) DNA?
1. DNA template.
2. Building block of DNA (dNTPs).
3. DNA copier (an enzyme).
4. 3’OH (primer).

9. Components: (1) DNA template
• The DNA sample you collect from a crime scene
or the one under investigation is the DNA template.
G A
C T
A
T
T
A
G
C
G
C
A
T
C
G
C
G
G
C
T
A
A
T
T
A
G
C
A
T
C
G
A
T
C
G
5’
5’
3’
3’
C TT A C C TG G CA T A C T G T G
5’3’
G AA T G G AC C GT A T G A C A C
5’ 3’
Each strand serves as a template for copying.
Remember complementary base-pairing!

10. Components: (2) dNTP (building blocks)
Do you remember? DNA is made of nucleotides!

11. Deoxyribonucleoside triphosphate (dNTP)
H
Four dNTPs serve as the building blocks of DNA
(dATP, dTTP, dGTP, dCTP)
Remember Nucleotides!
Components: (2) dNTP (building blocks)

12. Deoxyribonucleoside triphosphate (dNTP)
Why triphosphate?
For the energy required to for the phosphodiester
bond
+
H H
Components: (2) dNTP (building blocks)

13. Components: (3) DNA copier (polymerase)
• DNA polymerase is the DNA copier in the cell.
• Uses the dNTPs (DNA building blocks) to make
a complementary strand to the template.
• Uses the available 3’-OH of a previous
nucleotide and 5’phsphate from dNTP to form a
phosphodiester bond.

14. Components: (3) DNA copier (polymerase)
• Each time DNA Pol finds the correct
complementary dNTP and catalyzes the reaction
linking the new nucleotide.
Remember DNA Pol needs 3’-OH

15. Primers are short piece of polynucleotide
G
C T
A
T
T
A
G
C C TG G CA T A C T G T G
5’
5’
OH-3’
3’
In order for the DNA copying machine to work and
add nucleotides,
a 3’-OH needs to be available to form a
phosphodiester bond!
Components: (4) primer (3’ OH)

16. PCR Process
• Three steps are involved in PCR:
1. DNA template denaturation:
separation of the two strands of DNA.
2. Primers annealing: small
oligonucleotide attaches to each
separated strand providing the 3’OH
for DNA polymerase.
3. DNA polymerization (extension):
DNA polymerase extends the primers
on both strands and adds nucleotides.

17. PCR Process
1. DNA denaturation 2. Primers annealing 3. DNA extension

18. PCR cycles
1. DNA denaturation 2. Primers annealing 3. DNA extension
Temp(C)
94 C
55-65 C
72 C
What happens if we repeat this cycle many
times?
2 copies
4 copies
8 copies
16 copies
32 copies
Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5

19. PCR cycles
21 22 23 24 25
Exponential growth in the number of copies
generated.
The number of copies you get at the end of your
PCR will be 2#cycles (236 cycles = 68 billion copies)
2 copies
4 copies
8 copies
16 copies
32 copies
Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5

20. PCR – how many copies?
#DNAcopies
# cycles
Threshhold
Not enough DNA
template
No chemicals left in the
reaction tube

21. Problems!
There were some difficulties with this
system:
1.Three water-baths with three different
temperature.
2.DNA polymerase denatures at 94 C.

22. Problems!
DNA denature
(94 C)
Primer annealing
(55-65 C)
Extension
(72 C)
• The sample has to be transferred into multiple
water baths to accommodate the needed
temperature.

23. Problems!
DNA denature
(94 C)
Primer annealing
(55-65 C)
Extension
(72 C)
Adding
DNA Pol
DNA Pol
denatures
• DNA polymerase needs to be added in every
cycle because DNA polymerase denatures at
high temperature.

24. Improvement 1
• Using Thermus aquaticus (Taq) polymerase.
• Taq polymerase is heat stable and the cycles
can take place without the polymerase being
destroyed during the denaturation phase

25. Improvement 2
Replacing old machine (water baths) with a
thermocycler

26. To consider
• Length and GC content of your primer.
• Compatibility of your forward and reverse primers.
• Primer’s sequences do not complement each
other (primer dimer).
• Annealing temperature of both primers should be
the same.
• Length of the target DNA piece ( the longer the
target the longer the extension time).
• DNA polymerase, primers and other chemicals’
concentration should be precisely calculated.

27. What is DNA sequencing?
It is reading the letters of the book.
It is reading the exact nucleotide sequence of the
genome.

28. Sequencing methods available now!
1. Maxam and Gilbert chemical
degradation method (extinct).
2. Sanger sequencing (dideoxy
or chain termination method).
3. Illumina sequencing.
4. SOLiD sequencing.
5. Pyrosequencing.
6. Ion Torrent method.
7. Single molecule sequencing.

29. Why DNA sequencing?
• DNA sequencing can be considered the ultimate
characterization of gene(s) or fragment(s) of DNA.
• DNA Sequencing is used for:
• Mapping genomes
• Determining gene structure and thus function
• Detecting polymorphism (single nucleotide
polymorphism SNP)
• Analyzing genetic variation
• Predicting the possible product(s) of DNA
fragments
• Many purposes depending on the questions one is
asking

30. Sanger sequencing (the great method)
1.Fredrick Sanger has
developed a sequencing
method and received a
Noble prize for it.
2.Sanger sequencing method
is also called Chain
Termination Method and
Dideoxy sequencing
method.

31. Sanger sequencing (the great method)
• Employs:
• specific primers
• dNTPs
• ddNTPs
• DNA polymerase
• DNA template

32. DNA synthesis
+
H H
DNA synthesis requires the availability of a
3’-OH and energy

33. DNA synthesis
Difference in OH location in sugar and
consequences

34. DNA synthesis
The absence of OH group on the 3’ carbon of the
sugar blocks further addition of nucleotides

35. Sanger sequencing procedure
ddGTP ddCTP ddATP ddTTP
DNA Template
Polymerase
Excess dNTPs
Primer

36. 3’5’5’5’
G5’
G A5’
AG A5’
AG A T5’
AG TA TAG TA T5’
C C5’
AG CTA TAG CTA T T5’
C
Sanger sequencing procedure
AG TA TGAATTC3’ 5’
5’
G
5’
G A
5’
AG A
5’
AG A T
5’
AG TA T
AG TA T5’
C
C5’
AG CTA T
AG CTA T T5’
C

37. A T G C
+
3’
5’
• Analysis using high
resolution
polyacrylamide gel
electrophoresis.
• Fragments are
detected using
radioactive markers
and autoradiography.
A
C
T
G
G
T
C
A
A
T
C
G
A
T
C
G
T
A
Sanger sequencing procedure

38. Sanger sequencing - Gel

39. • Each dideoxy nucleotide is attached to a
florescent marker.
• At the end of each cycle, a laser beam can detect
the florescent marker and thus record the position
of the nucleotide.
Sanger sequencing - Automated

40. Sanger sequencing - Automated

41. Chromatogram - Automated

42. To know
Polymerase chain reaction
PCR
DNA template
dNTPs
ddNTPs
DNA polymerase
Taq polymerase
primer
3’-OH
denaturation
annealing
extension
DNA copies
2 #cycle
thermocycler
Sanger sequencing
Dideoxy sequencing
Chain termination sequencing
Polyacrylamide gel
Automated sanger sequencing
Capillary electrophoresis
chromatogram

43. Expectations
• You know PCR’s components and process.
• You know the phases of PCR and what
happens in each phase.
• You know how important it is for molecular
applications.
• You know DNA sequencing using Sanger
method.

44. For a smile