Pcr, rapd dan rflp

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