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Polymerase Chain Reaction

Kuliah Blok 9 FK Unsri 2014 oleh: Zen Hafy

• Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry

Polymerase Chain Reaction (PCR) • PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested. • The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.

Development…. • PCR work was first published (1985)using Klenow polymerase – unstable with heat • New enzyme had to be added manually at each step • Maximum length 400bp • Great idea – not very practical • First reports using DNA polymerase from Thermus aquaticus (1988) • Taq-polymerase (Saiki et al, 1988) from Yellow stone National Park hot springs • Developed automatic “thermocycler” programmable heat block

What all PCR Can Do ? • 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 (i.e., HIV, Vibrio sp., Salmonella sp. etc.) • Amplified fragments can act as genetic fingerprints

PCR Thermocycler

PROCEDURE …..

Nucleoside (dNT) Nucleotide (dNTP)

PCR Reagents • 1X Buffer – 10mM Tris-HCl, 50mM KCl

• MgCl2

– 1mM - 4mM (1.5mM)

• dNTPs – 200μM

• Primers – 100nM-1μM, 200nm (or less) for real time analysis

• DNA polymerase – Taq DNA polymerase is thermostable – 1-4 Units (1 unit)

• DNA (template DNA) – 10pg-1μg (20ng)

DNA

Primer

dNTP (dATP, dGTP,dCTP,dTTP)

Polymerase Chain Reaction

Initiation - Forming the Replication Eye Origin of Replication

5’

3’

3’

5’

3’

5’

5’

3’

3’

5’ 5’

5’

3’

3’

5’

3’

3’

5’ 5’

3’

Extension - The Replication Fork 5’ 3’

3’

5’ 3’

5’

5’

3’ 5’

Primase

Laging Strand Okazaki fragment

5’ RNA Primers

3’

Single strand binding proteins

5’

DNA Polymerase

5’ 3’ Helicase

Leading Strand

5’ 3’

How are the functions of replication achieved during PCR ??? Function

PCR

. Heat

ENZYMES



Melting DNA



Polymerizing DNA

. Taq Polymerase

•DNA pol



Providing primer

. Primers added to

•Primase

Joining nicks

. N/A as fragments



• Helicase •SSB proteins •Topoisomerase

the reaction mix

are short

•Ligase

Temperature

PCR Melting 94 oC 100

50

0

T i m e 3’

5’

5’

3’

Temperature

PCR 100

Melting 94 oC

50

0

T i m e 3’

5’

Heat 5’

3’

Temperature

100

Melting 94 oC

50

0

PCR

Melting 94 oC Extension Annealing oC 72 Primers 50 oC

T i m e 3’

5’ 5’

5’ 5’

3’

Temperature

PCR 100

Melting 94 oC

50

0

Melting 94 oC Extension Annealing 72 oC Primers 50 oC

T i m e 3’

5’

Heat 5’

5’

Heat 5’ 5’

3’

30x

Temperature

100

Melting 94 oC

50

0

PCR

Melting 94 oC Extension Annealing 72 oC Primers 50 oC

T i m e 3’

5’

5’

5’

5’

5’

5’

5’

5’

3’

30x

Temperature

100

50

0 3’

5’

5’

Melting 94 oC

PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC

T i m e

5’

5’ 5’

3’

Heat 5’

5’

Heat 5’

30x

Temperature

PCR 100

50

0 3’

5’

5’

Melting 94 oC Extension Annealing 72 oC Primers 50 oC

T i m e 5’

5’ 5’

Melting 94 oC

5’

3’

5’

5’

5’

5’

5’

5’

30x

Temperature

PCR 100

Melting 94 oC

50

0 3’

5’

5’

T i m e 5’

5’ 5’

Melting 94 oC Extension Annealing 72 oC Primers 50 oC

5’

3’

Fragments of defined length

5’

5’

5’

5’

5’

5’

30x

More Cycles = More DNA Size Number of cycles Marker 0 10 15 20 25 30

PCR Optimisation 1: Buffers • Most buffers have only KCl (50mM) and Tris (10mM) – Concentrations of these can be altered – KCl facilitates primer binding but concentrations higher than 50mM inhibit Taq • DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet P-40, Triton X-100 can be added to aid in the PCR reaction – Enhance specificity, but also can be inhibitory • Pre-mixed buffers are available

PCR Optimisation 2: MgCl2 • MgCl2: required for primer binding – MgCl2 affects primer binding, Tm of template DNA, product- and

primer-template

associations,

product

specificity, enzyme activity and fidelity – dNTPs, primers and template chelate and sequester the Mg ion, therefore concentration should be higher than dNTPs (as these are the most concentrated) – Excess magnesium gives non-specific binding – Too little magnesium gives reduced yield

PCR Optimisation 3: Primer Design • Specific to sequence of interest – Length 18-30 nucleotides • Annealing temperature 50oC-70oC – Ideally 58oC-63oC • GC content 40-60% • 3’ end critical (new strand extends from here) • GC clamp (G or C at 3’ terminus) • Inner self complementarity: – Hairpins <5, dimers <9 • 3’ complementarity: – <3-4 bases similar to other primer regions

In summary

• Primer length should not exceed 30 mer. • Tm, not more than 60 degree . • GC Content should be in the range of 40-60 % for optimum PCR efficiency. • Primers should end (3′) in a G or C, or CG or GC: this prevents “breathing” of ends and increases efficiency of priming.

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

Primer3 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

PCR Optimisation 4: Cycling Conditions • Denaturation: – Some Taq polymerases require initial denaturation (hot start)

• Annealing temperature: – ~ 5oC less than Tm of primers – Tm = 4(G + C) + 2(A + T)oC (or use of primer software) – Decrease in annealing temperature result in nonspecific binding – Increase in annealing temperature result in reduced yield

PCR Optimisation 5: Cycle Number • 25-40 cycles • Half-life of Taq is 30 minutes at 95oC • Therefore if you use more than 30 cycles at denaturation times of 1 minute, the Taq will not be very efficient at this point

Theoretical yield = 2n ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc eg. if you start with 100 copies after 30 cycles you will have 107, 374, 182, 400 copies

Variations of the PCR • • • • • • • • • • • • •

Colony PCR Nested PCR Multiplex PCR AFLP PCR Hot Start PCR In Situ PCR Inverse PCR Asymmetric PCR Long PCR Long Accurate PCR Reverse Transcriptase PCR Allele specific PCR Real time PCR

Types of PCR Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses primers external to the sequence of interest. The second PCR uses the product of the first PCR in conjunction with one or more nested primers to amplify the sequence within the region flanked by the initial set of primers.

Quantitative PCR: Product amplification w r t time, which is compared with a standard DNA. Hot start PCR: Used to optimize the yield of the desired amplified product in PCR and simultaneously to suppress nonspecific amplification.

Multiplex PCR •

Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers.

Reverse Transcriptase PCR • Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses. • First step of RT-PCR - "first strand reaction“-Synthesis of cDNA using oligo dT primers (37°C) 1 hr. • “Second strand reaction“-Digestion of cDNA:RNA hybrid (RNaseH)-Standard PCR with DNA oligo primers.

• Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.

Why real time PCR ? • QUANTITATION OF mRNA

– – – –

northern blotting ribonuclease protection assay in situ hybridization RT-PCR • • • •

most sensitive can discriminate closely related mRNAs technically simple but difficult to get truly quantitative results using conventional PCR

Real-Time PCR Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

• Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring (Real-Time). • Real-time PCR uses a fluorescent reporter signal

to measure the amount of amplicon as it is generated. This kinetic PCR allows for data

collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.

Real-time PCR advantages * amplification can be monitored real-time

* no post-PCR processing of products (high throughput, low contamination risk) * ultra-rapid cycling (30 minutes to 2 hours) * wider dynamic range of up to 1010-fold * requirement of 1000-fold less RNA than conventional assays (6 picogram = one diploid genome equivalent) * detection is capable down to a two-fold change * confirmation of specific amplification by melting curve analysis * most specific, sensitive and reproducible * not much more expensive than conventional PCR (except equipment cost)

Real-time PCR disadvantages

* Not ideal for multiplexing * setting up requires high technical skill and support * high equipment cost

* intra- and inter-assay variation * RNA liability * DNA contamination (in mRNA analysis)

Applications of PCR • Classification of organisms • Genotyping • Molecular archaeology • Mutagenesis • Mutation detection • Sequencing • Cancer research

• Detection of pathogens • DNA fingerprinting • Drug discovery • Genetic matching • Genetic engineering • Pre-natal diagnosis

Thank you THANK YOU

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