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POLYMERASE CHAIN

REACTION (PCR)

AMPLIFIKASI FRAGMEN DNA

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Background

Ability to generate identical high

copy number DNAs madepossible in the 1970s byrecombinant DNA technology(i.e., cloning).

Cloning DNA is time consumingand expensive (>>$15/sample).

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PCR, discovered in 1983 by Kary Mullis, enables theamplification (or duplication) of millions of copies of any DNAsequence with known flanking sequences.

Requires only simple, inexpensive ingredients and a couple hours.

DNA template

Primers (anneal to flanking sequences)

DNA polymerase

dNTPs

Mg2+

Buffer

Can be performed by hand or in a machine called a thermalcycler.

1993: Nobel Prize for Chemistry

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The polymerase chain reaction (PCR) can

selectively and rapidly amplify a given DNAsequence to large amounts

Usedincloning, sequencing, forensics, diagnosis

Specificprimers hybridize on each side ofthe DNA

sequence to be copied

Enzyme Taq DNA polymerase fromThermus

aquaticus resistantto high temperatures

Very sensitive canamplify a sequence presentin

very lowcopy number

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How PCR works:

1. Begins with DNA containing a sequence to be amplified and a pairof synthetic oligonucleotide primers that flank the sequence.

2. Next, denature the DNA at 94C.

3. Rapidly cool the DNA (37-65C) and anneal primers tocomplementary s.s. sequences flanking the target DNA.

4. Extend primers at 72C using a heat-resistant DNA polymerase

(e.g., Taq polymerase derived from Thermus aquaticus).

5. Repeat the cycle of denaturing, annealing, and extension 20-45times to produce 1 million (220)to 35 trillion copies (245) of thetarget DNA.

6. Extend the primers at 72C once more to allow incomplete

extension products in the reaction mixture to extend completely.

7. Cool to 4C and store or use amplified PCR product for analysis.

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Hot water bacteria:Thermus aquaticus

Taq DNA polymerase

Life at High Temperaturesby Thomas D. BrockBiotechnology in Yellowstone

1994 Yellowstone Association for Natural Sciencehttp://www.bact.wisc.edu/Bact303/b27

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Example thermal cycler protocol used in lab:

Step 1 7 min at 94C Initial Denature

Step 2 45 cycles of:

20 sec at 94C Denature20 sec at 64C Anneal1 min at 72C Extension

Step 3 7 min at 72C Final Extension

Step 4 Infinite hold at 4C Storage

BIOL 362 samples processed in:MJ Research DNA Engine Dyad

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Fig. 7.23

Denature

Anneal PCRPrimers

Extend PCRPrimersw/Taq

Repeat

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10_27_1_PCR_amplify.jpgThe polymerase chain reaction usedtoamplify a specific

DNA sequence with cyclical changes intemperature

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10_27_2_PCR_amplify.jpg

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PCR applications:

1) The methodofchoice forcloning relatively short

DNA sequences (under10,000nts) can use to get

genomicclone orcDNA clone

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10_28_PCR_clones.jpg

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PCR applications:

1) The methodofchoice forcloning relatively short

DNA sequences (under10,000nts) can use to get

genomicclone orcDNA clone

2) Candetectinfectious pathogens atvery earlystages

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10_29_PCR_viral.jpgUsing PCR todetectaviral genome inadropofblood

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53

35

5

3

3

5

Melt template, then rapidly cool

* some primers will anneal to complementary sequence

5 3

53

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53

35

5

3

3

5

Melt template, then rapidly cool

* some primers will anneal to complementary sequence

Add DNA polymerase

* provide substrate + time to extend

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53

35

5

3

3

5

Melt template, then rapidly cool

* some primers will anneal to complementary sequence

Add DNA polymerase

* provide sunstrate + time to extend

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These 3 steps constitute 1PCR cycle, which will be repeated

many times (usually 25-30)

1) melt template

2) anneal oligonucleotide primers

3) extend with DNA polymerase

If ever confused about how PCR works

* draw out first three cycles

25-30x

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53

35

5

3

3

5

First cycle

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53

35

5

3

3

5

First cycle

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53

35

5

3

3

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First cycle

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Second Cycle

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Second Cycle

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Second Cycle

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Third cycle

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Third cycle

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Third cycle

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Third cycle

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From 3rd cycle onwards this species will predominate

Once it gets going truly exponential growth

amplification = 2n

(n = # cycles)

So, 30-35 cycles, 10 billion-fold amplification

- in reality, will never get this much

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Limitations finite amounts of

* dNTPs

* primers

* DNA pols

Exhaustion after 30

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AKUMULASI EKSPONENSIAL

FRAGMEN TERAMPLIFIKASI

Setelah 30 siklus

2pangkat28 = 268 345 456fragmen

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Good Primers Characteristic

A melting temperature (Tm)inthe range of

52 C to65 C

Absence ofdimerizationcapability Absence ofsignificant hairpinformation

(>3 bp)

Lackofsecondary priming sites Low specific binding atthe 3' end (ie.

lower GC contenttoavoidmispriming)

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UniquenessThere shall be one and only one target site in the template DNAwhere the primer binds, which means the primer sequence shall beunique in the template DNA.

There shall be no annealing site in possible contaminant sources,such as human, rat, mouse, etc. (BLAST search against

corresponding genome)

Primer candidate 1 5-TGCTAAGTTG-3

Primer candidate 2 5-CAGTCAACTGCTAC-3

TGCTAAGTTG CAGTCAACTGCTAC

Template DNA5...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3

NOT UNIQUE!

UNIQUE!

TGCTAGTTG

A

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Length

Primer length has effects on uniqueness andmelting/annealing temperature. Roughly speaking, thelonger the primer, the more chance that its unique; the

longer the primer, the higher melting/annealingtemperature.

Generally speaking, the length of primer has to be atleast 15 bases to ensure uniqueness. Usually, we pick

primers of17-28

bases long. This range varies basedon if you can find unique primers with appropriateannealing temperature within this range.

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PANJANG PRIMER

Panjang primer8

4 pangkat8 = 65.536pb

Ukurankromosom 3.000.000kb ada 46.000

kemungkinan situs

Panjang primer17

= 17.179.869.184 pb diharapkan hanyamenempel pada1 situs

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Base Composition

Base composition affects hybridization specificity andmelting/annealing temperature.

Random base composition is preferred. We shall avoid long (A+T)and (G+C) rich region if possible.

Usually, average (G+C) content around 50-60% will give us theright melting/annealing temperature for ordinary PCR reactions, andwill give appropriate hybridization stability. However,melting/annealing temperature and hybridization stability areaffected by other factors. Therefore, (G+C) content is allowed tochange.

Template DNA5...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3

TGCCC G GCCCGATCATGCT