Implikasi REPLIKASI DNA DAN PCR (Polymerase Chain

(1)

Implikasi REPLIKASI

DNA DAN PCR

(

Polymerase Chain

Reaction

)

(2)

What is PCR?

It was invented in 1983 by Dr. Kary

Mullis, for which he received the Nobel

Prize in Chemistry in 1993.

PCR is an exponentially progressing

synthesis of the defined target DNA

(3)

What is PCR? :

Why

“

Polymerase

”

?

(4)

What is PCR? :

Why

“

Chain

”

?

It is called “chain” because the

(5)

History

• The Polymerase Chain Reaction

(PCR) was not a discovery, but rather

an invention

• A special DNA polymerase (

Taq

) is

used to make many copies of a short

length of DNA (100-10,000 bp)

(6)

What PCR Can Do

• PCR can be used to make many copies of any DNA that is supplied as a template

• Starting with one original copy an almost infinite number of copies can be made using PCR

• “Amplified” fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis

• Defective genes can be amplified to diagnose any number of illnesses

• Genes from pathogens can be amplified to identify them (ie. HIV)

(7)

How PCR Works

• PCR is an artificial way of doing DNA

replication

• Instead of replicating all the DNA

present, only a small segment is

replicated, but this small segment is

replicated many times

• As in replication, PCR involves:

–Melting DNA

–Priming

(8)

Initiation

-Forming the

Replication Eye

3’ 5’ 3’ 5’ 5’ 5’ 3’ 3’

Origin of Replication

(9)

Leading Strand Leading Strand Laging Strand Laging Strand 3’ 5’ 3’ 5’

Extension - The Replication Fork

(10)

Functions And Their

Associated Enzymes

è Ligase

• Joining nicks

è DNA

Polymerase

• Polymerizing

DNA

è Primase

• Providing primer

Enzyme

Function

è Helicase

è SSB Proteins

è Topisomerase

(11)

Components of a PCR

Reaction

• Buffer (containing Mg

++

)

• Template DNA

• 2 Primers that flank the fragment of

DNA to be amplified

• dNTPs

(12)

PCR

The cycling react ions :

There are t hree m aj or st eps in a PCR, w hich are repeat ed for 20 t o 40 cycles. This is done on an

aut om at ed Th e r m o Cycle r, w hich can heat and cool t he react ion t ubes in a very short t im e.

Denat urat ion at around 94°C :

(13)

Annealing at around 54°C :

Hydrogen bonds are const ant ly form ed and broken bet w een t he single st randed

prim er and t he single st randed t em plat e. I f t he prim ers exact ly fit t he t em plat e, t he hydrogen

bonds are so st rong t hat t he prim er st ays at t ached

Ext ension at around 72°C :

The bases ( com plem ent ary t o t he t em plat e) are coupled t o t he prim er on t he 3' side ( t he

polym erase adds dNTP's from 5' t o 3', reading t he t em plat e from 3' t o 5' side, bases are added

(14)

PCR

(15)

PCR

Melting 94

o C

Temperature

100 50 0

T i m e

5’

3’

(16)

PCR

Melting 94

o C

Temperature

100 50 0

T i m e

3’

5’

3’

(17)

PCR

Melting 94 o C Annealing Primers 50 o C Extension 72 o C

Temperature

100 50 0

T i m e

(18)

PCR

Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C

Temperature

100 50 0

T i m e

(19)

PCR

Temperature

100 50 0

T i m e

(20)

PCR

Temperature

100 50 0

T i m e

(21)

PCR

Temperature

100 50 0

T i m e

(22)

Fragments of

defined length

PCR

Melting 94 o_C

Annealing Primers

50 o_C

Extension 72 o_C

Temperature

100

0 50

T i m e

(23)

Theoretical Yield Of PCR

Theoretical yield = 2

n

x

y

Where y = the starting

number of copies and

n = the number of thermal cycles

= 107,374,182,400

If you start with 100 copies, how many copies are

made in 30 cycles?

2

n

x y

= 2

30

x 100

(24)

(25)

How The Functions Of Replication

Are Achieved During PCR

è N/A as fragments

are short

• Joining nicks

è Primers are

added to the

reaction mix

• Providing primer

è

Taq

DNA

Polymerase

• Polymerizing

DNA

PCR

Function

è Heat

(26)

PCR and Contamination

The m ost im port ant considerat ion in PCR is cont am inat ion

Even t he sm allest cont am inat ion wit h DNA could affect am plificat ion

For exam ple, if a t echnician in a crim e lab set up a t est react ion

( wit h blood from t he crim e scene) aft er set t ing up a posit ive cont rol react ion ( wit h blood from t he suspect ) cross cont am inat ion bet w een t he sam ples could result in an erroneous incrim inat ion, even if t he t echnician changed pipet t e t ips bet ween sam ples. A few blood cells could volit ilize in t he pipet t e, st ick t o t he plast ic of t he pipet t e, and t hen get ej ect ed int o t he t est sam ple

(27)

Optimizing PCR protocols

While PCR is a very pow erful t echnique, oft en

enough it is not possible t o achieve opt im um result s w it hout opt im izing t he prot ocol

Crit ical PCR param et ers:

- Concent rat ion of DNA t em plat e, nucleot ides, divalent cat ions

( especially Mg2+ _{) and polym erase}

- Error rat e of t he polym erase (Taq, Vent exo, Pfu)

- Prim er design

(28)

(29)

DNA Sequencing

Sequencing methods

- The process of determining the order of the nucleotide bases along a DNA strand is called DNA sequencing

- In 1977 two separate methods for sequencing DNA were developed: the chain termination method or cycle sequencing (Sanger et al.) and the chemical degradation method or Maxam-Gilbert sequencing (Maxam and Gilbert)

- Both methods were equally popular to begin with, but, for many

reasons, the cycle sequencing method is the method more commonly used today

- This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be

(30)

Cycle Sequencing

Concept: If we know the distance of each type of base from a known origin, then it is possible to deduce the sequence of the DNA.

Obt aining t his inform at ion is concept ually quit e sim ple. The idea is t o cause a t erm inat ion of a grow ing DNA chain at a know n base ( A,G,C or T) and at a know n locat ion in t he DNA

I n pract ice, chain t erm inat ion is caused by t he inclusion of a sm all am ount of a single dideoxynucleot ide base in t he m ixt ure of all four

(31)

Deoxy versus dideoxy

(32)

DNA synthesis

(33)

Metode dalam sekuensing:

1. Metode Sanger (1977): prinsip dasar: dideoksi OHÆH

O- P O O CH2 H H H H

O- P O O

CH2

H O

H A : T

H H H H H G: C Diseoksinukleotida Sintesa berhenti

(34)

DNA Sequencing

(35)

DNA Sequencing

Cycle sequencing chain t erm inat ion

Taken from :

(36)

DNA Sequencing

The separat ion of t he sequencing fragm ent s

(37)

(38)

DNA Sequencing

Sequencing syst em s

LI COR DNA 4300

(39)

A

(40)

DNA Sequencing

Snapshot s of t he det ect ion of t he fragm ent s on t he sequencer

(41)

DNA Sequencing

C

h

ro

m

a

to

g

ra

m

f

il

(42)

DNA Sequencing

(43)

Maxam dan Gilbert (1977):

Prinsip: degradasi struktur kimia DNA

Gambar 1. Sekuensing dengan metodeMaxam-Gilbert

5‘ 3‘

32_P

G (A+G) C (C+T) A>C

Tambahkan senyawa pendegradasi

ACACTGAACGTTCATGTCGA………….. ACACTGAACGTTCATGTCGA………….. ACACTGAACGTTCATGTCGA………….. 32_P 32_P 32_P me me me ACACT ACACTGAAC ACACTGAACGTTCAT 32_P 32_P 32_P

Penghilangann senyawa modifikasi dan hidrolisa ikatan fosfat dengan

piperidine