PCR, RAPD dan

RFLP
Polymerase Chain
Reaction

PCR
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
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++.
 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.
 PCR
30x
100 Melting Melting

Temperature
94 oC Extension 94 oC
Annealing 72 oC
Primers
50 50 oC

0
T i m e 3’ 5’
3’ 5’

3’ 5’
5’ 5’
3’ 5’
5’

3’ 5’
5’ 3’ 5’
5’ 3’
5’ 5’
5’ 3’

5’ 3’
5’ 3’
PCR

Temperature
100 Melting
94 oC

50

0
T i m e

3’ 5’
5’ 3’
PCR

Temperature
100 Melting
94 oC

50

0
T i m e
3’ 5’

Heat

5’ 3’
PCR

Temperature
100 Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’
5’

5’
5’ 3’
PCR

Temperature
100 Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’

Heat
5’

5’

Heat
5’
5’ 3’
PCR

Temperature
100 Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’
5’

5’
5’

5’
5’

5’
5’ 3’
PCR

Temperature
100 Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’
5’
5’ T i m e
5’
5’
5’ 3’

Heat
5’

5’

Heat
5’
PCR

Temperature
100 Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’
5’
5’ T i m e
5’
5’ 5’
5’ 3’

5’
5’

5’
5’

5’
5’
PCR

Temperature
100 Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’
5’
5’ T i m e
5’ 5’ 5’
5’ 3’

5’

Fragments of 5’

defined length
5’ 5’

5’
5’
DNA Between The Primers Doubles With Each
Thermal Cycle
Number
1 2 4 8 16 32 64

0 1 2 3 4 5 6
Cycles
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.
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
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
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"
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
Specific Primers : Primers
designed from already known
DNA sequences (genes)
RAPD
Random Amplified
Polymorphic DNA

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

Template
DNA

Primers point away

from each other, so
amplification
won’t happen
 RAPD

Template
DNA

Primers point in
the same direction,
so amplification
won’t happen
 RAPD

Template
DNA

> 2,000 bases

Primers too far

apart, so
amplification
won’t happen
RAPD

Template
DNA

Primers are just

100 - 1,500 bases
the right
distance apart,
so fragment is
amplified
Separated RAPD Fragments 4mM MgCl2 4mM MgCl2 2mM MgCl2
1.2 U Taq 0.6 U Taq 1.2 U Taq
5 pM OPA-16 10 pM OPA-16 10 pM OPA-16
RAPD reactions Which variable
were run in groups M 2 3 4 5 6 7 8 9 10 M has the greatest
of 3 using the same impact on
template and primer, fragment
but varying patterns?
Magnesium,
polymerase and
Lowering
primer
Magnesium ion
concentrations
concentration
results in loss of
the largest
Normal fragment visible
concentrations are in lanes 2-7
shown in yellow
text. M = A size
standard
Restriction Fragment
Length Polymorphism

RFLP
Recognizing/producing
polymorphism caused by
differential recognition
site of restriction enzyme
on DNA sequence
 A single nucleotide change can
make a difference

Wild-type allele AGATCT

TCTAGA

Restriction site

Mutant allele
AGAGCT
TCTCGA

Not a restriction site

RFLP-determination

 Differences in DNA-sequence
between the two parents ( due to
mutations )
 Differences in restriction - enzym
sites
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
Dominant vs Co-dominant

A B C

A B C
A=AA B=AB C=BB
The laboratory steps involved in
RFLP detection

 Isolation of DNA
 Restriction digestion and gel
electrophoresis
 DNA transfer by Southern blotting
 DNA hybridisation
Southern Blotting
Restriction sites
GAAATC
No EcoRI site
CTTTAG

A B C
Parent 1

A D E C
Parent 2

GAATTC
EcoRI site
CTTAAG
Restriction sites
A B C
Parent 1
probe
A D E C
Parent 2
probe

Probe recognizes complementary

sequence
Probe has a color label or is radio-active
 A B C B
Parent 1
A D E C
Parent 2 E
probe C C

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

D
Separation with gel electrophoresis;
A A
many many fragments
 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
mixture

Transfer solution

RNA

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?