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 ProcessPCR 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.
PCRMelting
94 oC
Melting
94 oC
AnnealingPrimers
50 oC
Extension
72 oCTe
mpe
ratu
re
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’
PCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
5’3’
3’5’
PCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’
Heat
PCRMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’5’
5’
Melting94 oC
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
Heat
Heat
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
Fragments of defined length
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’ 5’
5’
5’
5’
5’
5’
5’
5’
DNA Between The Primers Doubles With Each Thermal Cycle
0Cycles
Number1
3
8
2
4
1
2
4
16
5
32
6
64
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 Tm5oC
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 AARY= PyrimidineN= any baseR= purine
Specific Primers : Primers designed from already known DNA sequences (genes)
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
RAPD1. Buffer (containing Mg++)
- usually high Mg++ concentrations are used lowering annealing stringency
2. Template DNA3. 1 short primer (10
bases)not known to anneal to any specific part of the template DNA
4. dNTPs5. Taq DNA Polymerase
PCR1. Buffer (containing
Mg++)
2. Template DNA3. 2 Primers that flank
the fragment of DNA to be amplified
4. dNTPs5. Taq DNA Polymerase
(or another thermally stable 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
Primers too far apart, so amplification won’t happen
> 2,000 bases
Template DNA
Primers are just the right
distance apart, so fragment is
amplified
100 - 1,500 bases
RAPD
MM 2 3 4 5 6 7 8 9 10
Separated RAPD Fragments4mM MgCl2
1.2 U Taq5 pM OPA-16
4mM MgCl2
0.6 U Taq10 pM OPA-16
2mM MgCl2
1.2 U Taq10 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?
Restriction Fragment Length Polymorphism
RFLP
Recognizing/producing polymorphism caused by differential recognition site of restriction enzyme on DNA sequence
AGATCTWild-type allele
Mutant allele
TCTAGA
A single nucleotide change can make a difference
AGAGCT
TCTCGA
Restriction site
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 diploidTwo 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
B=AB C=BB
B CA
B C
A=AA
A
Dominant vs Co-dominant
The laboratory steps involved in RFLP detection
Isolation of DNARestriction digestion and gel
electrophoresisDNA transfer by Southern blottingDNA hybridisation
Southern Blotting
Restriction sites
B C
A D E C
A
Parent 2
Parent 1
GAATTCCTTAAG
GAAATCCTTTAG
No EcoRI site
EcoRI site
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
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
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
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
RNAmixture
Transfer solution