Mata kuliah tentang PCR, RAPD, dan RFLP.Cari lebih banyak lagi di: http://muhammadhabibielecture.blogspot.com/2015/02/materi-kuliah-semester-4.html

1. PCR, RAPD dan
RFLP

2. Polymerase Chain
Reaction
PCR

3. The polymerase chain
reaction(PCR) is to used to
amplify a sequence of DNA
using a pair of primers each
complementary to one end
of the DNA target sequence

4. The PCR cycle
• Denaturation: The target DNA (template) is
separated into two strands by heating to 95℃
• Primer annealing: The temperature is
reduced to around 55℃ to allow the primers to
anneal.
• Polymerization (elongation, extension):
The temperature is increased to 72℃ for optimal
polymerization step which uses up dNTPs and
required Mg++.

5. The PCR Process
PCR works like this:
– DNA and two primers are combined in a salt solution
with dNTPs and a heat stable DNA polymerase
enzyme
– The primers match some sequence in the target DNA
– The solution is rapidly heated to DNA denaturing
temperatures (~95°C) and cooled to a temperature
where the polymerase can function
– Each thermal cycle generates copies of the sequence
between the primers, so the total number of fragments
amplifies in an exponential fashion: 2, 4, 8,16, 32, 64,
etc.

6. PCR
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
5’3’
3’5’
3’5’
5’
5’3’
5’
3’5’
5’
5’
5’
5’3’
3’5’
3’5’
5’3’
5’3’
5’

7. PCR
Melting
94 oC
Temperature
100
0
50
T i m e
5’3’
3’5’

8. PCR
Melting
94 oC
Temperature
100
0
50
T i m e
3’5’
5’3’
Heat

9. PCR
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
3’5’
5’3’
5’
5’
Melting
94 oC

10. PCR
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’

11. PCR
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’

12. PCR
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
Heat
Heat

13. PCR
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

14. Fragments of
defined length
PCR
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’ 5’
5’
5’
5’
5’
5’
5’
5’

15. DNA Between The Primers Doubles With Each
Thermal Cycle
0
Cycles
Number
1
3
8
2
4
1
2
4
16
5
32
6
64

16. Template
•Any source of DNA that provides
one or more target molecules can
in principle be used as a template
for PCR
•Whatever the source of template
DNA, PCR can only be applied if
some sequence information is
known so that primers can be
designed.

17. Primers
• PCR primers need to be about 18 to 30
nt long and have similar G+C contents
so that they anneal to their
complementary sequences at similar
temperatures.They are designed to
anneal on opposite strands of the
target sequence
• Tm=2(a+t)+4(g+c): determine
annealing temperature. If the primer is
18-30 nt, annealing temperature can be
Tm5oC

18. Primer Design Rules
• primers should be at least 15 base pairs long
• have at least 50% G/C content
• anneal at a temperature in the range of 50-65
degrees C
• Usually higher annealing temperatures (Tm)
are better (i.e. more specific for your desired target)
• forward and reverse primer should anneal at
approximately the same temperature

19. Primer Problems
• primers should flank the sequence of interest
• primer sequences should be unique
• primers that match multiple sequences will give
multiple products
• repeated sequences can be amplified - but only if
unique flanking regions can be found where
primers can bind
• primers can have self-annealing regions within
each primer (i.e. hairpin and foldback loops)
• pairs of primers can anneal to each other to form
the dreaded "primer dimers"

20. Degenerate primers: an oligo
pool derived from protein sequence.
E.g. His-Phe-Pro-Phe-Met-Lys can
generate a primer
5’-CAY TTY CCN TTY ATG AAR
Y= Pyrimidine
N= any base
R= purine

21. Specific Primers : Primers
designed from already known
DNA sequences (genes)

22. Random Amplified
Polymorphic DNA
RAPD

23. Recognizing/producing
polymorphism caused by
differential amplification
of DNA sequence

24. History
 Shortly after Kary Mullis invented the
Polymerase Chain Reaction (PCR) it was
realized that short primers would bind to
several locations in a genome and thus could
produce multiple fragments
 Williams et al. (1990) developed Random
Amplified Polymorphic DNA (RAPD) a technique
using very short 10 base primers to generate
random fragments from template DNAs
 RAPD fragments can be separated and used as
genetic markers or a kind of DNA fingerprint

25. Components of a PCR and
RAPD Reactions
RAPD
1. Buffer (containing Mg++) -
usually high Mg++
concentrations are used
lowering annealing
stringency
2. Template DNA
3. 1 short primer (10 bases)not
known to anneal to any
specific part of the template
DNA
4. dNTPs
5. Taq DNA Polymerase
PCR
1. Buffer (containing Mg++)
2. Template DNA
3. 2 Primers that flank the
fragment of DNA to be
amplified
4. dNTPs
5. Taq DNA Polymerase (or
another thermally stable
DNA polymerase)

26. Modifying Thermal
Cycling
 Two modifications made to typical thermal
cycling when RAPD is being done:
1. Annealing temperatures are generally very
low, around 36 oC - This allows very short
primers to anneal to template DNA
2. More thermal cycles are used, typically 45 -
This compensates for the inefficiency which
results from using such short primers.

27. RAPD
Template
DNA
 Primer binds to many locations on the template
DNA
 Only when primer binding sites are close and
oriented in opposite direction so the primers point
toward each other will amplification take place

28. RAPD
Template
DNA
Primers point away
from each other, so
amplification
won’t happen

29. RAPD
Template
DNA
Primers point in
the same direction,
so amplification
won’t happen

30. RAPD
Template
DNA
Primers too far
apart, so
amplification
won’t happen
> 2,000 bases

31. Template
DNA
Primers are just
the right
distance apart,
so fragment is
amplified
100 - 1,500 bases
RAPD

32. MM 2 3 4 5 6 7 8 9 10
Separated RAPD Fragments4mM MgCl2
1.2 U Taq
5 pM OPA-16
4mM MgCl2
0.6 U Taq
10 pM OPA-16
2mM MgCl2
1.2 U Taq
10 pM OPA-16
Normal
concentrations are
shown in yellow
text. M = A size
standard
Lowering
Magnesium ion
concentration
results in loss of
the largest
fragment visible
in lanes 2-7
RAPD reactions
were run in groups
of 3 using the same
template and primer,
but varying
Magnesium,
polymerase and
primer
concentrations
Which variable
has the greatest
impact on
fragment
patterns?

33. Restriction Fragment
Length Polymorphism
RFLP

34. Recognizing/producing
polymorphism caused by
differential recognition
site of restriction enzyme
on DNA sequence

35. AGATCT
Wild-type allele
Mutant allele
TCTAGA
A single nucleotide change can
make a difference
AGAGCT
TCTCGA
Restriction site
Not a restriction site

36. RFLP-determination
 Differences in DNA-sequence
between the two parents ( due to
mutations )
 Differences in restriction - enzym
sites

37. Dominant vs Co-dominant
 Most organisms we study are diploid
 Two sets of chromosomes
Co-dominant:
the marker on both chromosomes is
visible and distinguishable
Dominant: the marker is present and you
can not see whether is coming from both
chromosomes or from only one

38. B=AB C=BB
B CA
B C
A=AA
A
Dominant vs Co-dominant

39. The laboratory steps involved in
RFLP detection
 Isolation of DNA
 Restriction digestion and gel
electrophoresis
 DNA transfer by Southern blotting
 DNA hybridisation

40. Southern Blotting

41. Restriction sites
B C
A D E C
A
Parent 2
Parent 1
GAATTC
CTTAAG
GAAATC
CTTTAG
No EcoRI site
EcoRI site

42. Restriction sites
B C
A D E C
A
Parent 2
Parent 1
probe
Probe recognizes complementary
sequence
Probe has a color label or is radio-active
probe

43. A
C C
A
D
E
B C
A D E C
A
Parent 2
Parent 1
probe
Separation with gel electrophoresis;
smaller fragments run faster
B

44. A
C C
A
D
E
B C
A D E C
A
Parent 2
Parent 1
probe
Separation with gel electrophoresis;
many many fragments
B

45. Question:
You are using Northern blotting to analyze two mRNA samples derived from fibroblasts and
hepatocytes. What will you see if you use a probe made from exon EIIIB of the fibronectin gene?
What about using a probe made from the exon next to EIIIB?
Detection of alternative splicing by Northern blotting
•Northern blotting can be used to detect specific RNAs in complex mixtures.
•Southern blotting detects specific DNA fragments.
•Western blotting (immunoblotting) detects specific proteins with antibodies.
RNA
RNA
mixture
Transfer solution