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Polymerase Chain Reaction(PCR)

Dr Cath ONeill

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PCR

A means of selectively amplifying a

particular segment of DNA in vitro. The

DNA to be amplified can be a whole geneor part of a gene. Often the DNA to be

amplified is part of a large and complex

mixture.

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Components of typical PCR

reaction Templatethe DNA that contains the target sequence

you want to amplify (1.5-100ng)

Primerssynthetic oligonucleotide whose sequencematches a region flanking the target you want to amplify

and primes the manufacture of a new strand by DNApolymerase (50-100pmoles of each primer)

Buffer(supplied with the enzyme but typically containsTris/HCl (10-50mM), KCl (50mM), Triton-x-100 (0.1%)

Magnesium chloride(0.5-5mM) dNTPs(deoxynucleotides) (50-200uM of each dNTP)

DNA polymerase, usually Taq polymeraseHeat stablepolymerase (0.5-1u)

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Which bits of DNA get specifically

amplified?

The segment of DNA to be amplified is

flanked by two primers:

e.g.

5caatttggatagtagccgtatcgatgcgtagtcagatgaggtga

ccagtggatgacgattgacattaagtgaacggtgacccaaaagt

gacgatagacagttgacaaagttgacagtagacgatagacaa

gttgtttggaccaaagtgacacgtgacgttttgcaaacgtgacgtgaacgttgacacagtgac3

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3'

3'

3'

3' 5'

5'

5'

5'

Polymerase Chain Reaction

Target DNA

Denaturation

Extension of

primers

Annealing of

primers

Single-strandedDNA

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Strand separation

Anneal

primers

Extend

primers

95C

50-60C

72C

The PCR reaction has three stages

x~30

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PCR is an exponential reactionCYCLE NUMBER AMOUNT OF DNA

0 1

1 22 4

3 8

4 16

5 32

6 64

7 128

8 256

9 512

10 1,024

11 2,048

12 4,096

13 8,192

14 16,384

15 32,768

16 65,536

17 131,072

18 262,144

19 524,288

20 1,048,576

21 2,097,152

22 4,194,304

23 8,388,608

24 16,777,216

25 33,554,432

26 67,108,864

27 134,217,728

28 268,435,456

29 536,870,912

30 1,073,741,824

31 1,400,000,000

32 1,500,000,00033 1,550,000,000

34 1,580,000,000

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AMOUNTOFDNA

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PCR in detail

only two

partially double

strandedproducts are

produced

denature

Anneal primers

Extend primers

Cycle 1

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Round 2four

partially double

stranded DNAs

are formedbut

not the target

sequence

Cycle 2

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Products from round 2

Six partially double strandedproducts are formed plus two

copies of the target sequence.

At 30 cycles there are

1,073,741,764 target copies

(~1x109). There are also 60

other DNA copies!!

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PCR

Gel

electrophoresis

2-3 hours!!

The end result

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Checking its worked!

Checked by agarose gel electrophoresis

Is the product the expected size? Is there more than one product?

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Optimising the reaction

From thisTo this!!

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Optimising the Reaction

Denaturation

Primers can anneal non-specifically during

first round of denaturation.

Polymerase could extend the primers

Use Hot start PCR

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Optimising the Reaction

Annealing

The annealing temperature (Tm) is one of the most vital parameters in

a PCR reaction.

Primers have a calculated Tm :

Tm=4(G+C)+2(A+T)0

C

Annealing temperature chosen depends directly on the length and

composition of the primer

Too low a Tm results in non-specific priming whilst too high affects yield

as the likelihood of annealing is reducedUsuallystart with a Tm 50C below the lowest Tm of the primer pair

Annealing happens quickly - ~30sec! Longer annealing times may

produce unwanted products

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Annealing

Primer design is vital!!

Primers ideally 18-22 bases long

G/C content should be 45-55% The annealing temperature (Tm) should

be within 10C of each other. In practice

Tms of 50-80

o

c are common. The 3 most base should be C or G

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Designing Primers

Many web-based design programs

Things to watch out for:

Primers that from hairpins

5 GTTGACTTGATA

TGAACTCT-3

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Optimising the reaction

Mg2+concentrationMg2+is required for:

a)Template-primer stability

b)Activity and fidelity of Taq polymerase

However, dNTPs, template and primers all bind the cation

and limit its availability.

Usual to titrate the Mg2+ concentration in steps of 0.5mM

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Elongation time and temperature

Optimal temperature for Taq is 720C

Will extend at the rate of up to100bases/second

Rule of thumb: 1kb requires about 1min ofextension time

Paradoxhow can primers which anneal atan optimum temperature be elongated at aconsiderably higher temperature??

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Cycle Number?

Conc of

product

Cycle number

Increasing the cycle

number above ~35 has little

positive effect. The plateau

is reached when:

- reagents deleted

- polymerase damaged

Plateau

effect

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Other things you might find in a

PCR reactionHelix Destabilisers

Dimethyl sulphoxide

Dimethyl formamide

Urea

Thought to lower the Tm of the target DNACare- can also decrease activity of Taq!!

Additives

Glycerolimproves amplification of GC-rich sequences

Polyethyleneglycoloften used when template concentration is low asit promotes macromolecular association

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Equipment considerations

Thin walled tubes are used for most PCR

applications

Can be bought in 0.2,0.5 or lml format

PCR blocks are also available that take

plates (96well up to 384well)

If possible use a block with a heated lid

Peltier blocks are best because of the fast

heating and cooling (30C per second)

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PCR is extremely sensitive

ALWAYS run negative control!!!

Contamination by unwanted DNA can be a

problem

Gloves

Filtered tips

Dedicated areas (UV cabinet)

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Whats Wrong With

Agarose Gels?

* Poor precision

* Low sensitivity

* Short dynamic range < 2 logs* Non-automated

* Size-based discrimination only

* Results are not expressed as numbers

* Ethidium bromide staining is not very quantitative

ABI: Real-Time PCR vs Traditional PCR (www)

5 copies 50

copies

http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdfhttp://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

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Fluorescence-based probes

coupled with suitable

instrumentation have

revolutionised quantitative PCR

Real-Time PCRReal-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

CYCLE NUMBER AMOUNT OF DNA

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0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AMOUNT

OFDNA

1

10

100

1000

10000

1000001000000

10000000

100000000

1000000000

10000000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AMOUNTO

FDNA

0 1

1 2

2 4

3 8

4 16

5 32

6 64

7 1288 256

9 512

10 1,024

11 2,048

12 4,096

13 8,192

14 16,384

15 32,768

16 65,536

17 131,072

18 262,144

19 524,288

20 1,048,576

21 2,097,152

22 4,194,304

23 8,388,608

24 16,777,21625 33,554,432

26 67,108,864

27 134,217,728

28 268,435,456

29 536,870,912

30 1,073,741,824

31 1,400,000,000

32 1,500,000,000

33 1,550,000,000

34 1,580,000,000

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Data From a Real-Time PCR Experiment

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Log Plot of Real Data

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The Log Plot is Linear between 20 and 1500 Units

of Fluorescence

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The Threshold Cycle (Ct)

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Threshold Cycle

threshold cycle or the CTvalue isthe cycle at which a significant

increase in fluoresecnce is first

detectedCTco-incides with the linear phase

of amplification

it is the parameter used forquantitation

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So how do you make the PCR

products fluoresce??

e.gs of chemistry:

DNA Binding dyes (e.g. sybr green)

Hydrolysis probes

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Dye Incorporation Assay

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* Assays that do not require specificity of probe based assays.

Detection of 1000s of molecules

* General screening of transcripts prior to moving to probebased assays

* When the PCR system is fully optimized -no primer dimers or

non-specific amplicons, e.g. from genomic DNA

When to choose SYBR Green

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Hydrolysis probe (Taqman)

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Instrumentation

Instruments on the market:

1) ABI Prism 7900 (Perkin Elmer) (Taqman)can be used forassays based on DNA binding dyes, Molecular beacons,

Hydrolysis probes.

2) Lightcycler (Roche)can be used with any chemistry

iFluorescence is measured in real-time

3) MyiQ cycler (Biorad)optical module that fits onto their

regular PCR machine - can use any chemistry

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Real-Time PCR for Cancer

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Real-Time PCR for CancerDiagnostics

Mutation Detection Using

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Mutation Detection UsingLightcylcer Technology

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Multiplex PCRi.e.Screening for multiple genes

simultaneously

Advantageslower reagent costs

speed

Particularly good if tissue is

limited

Disadvantageslimited availability of

fluorescent probes

Depends on which Instrument is

available

Advantages of PCR-Based

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Advantages of PCR-BasedDiagnostics

-all closed tube systems, minimises hands on time

-- minimal contamination

-- entire process is automated

- Speed

-Cost effective

-- assays easily standardised

-Detection down to a 2x change

-Not much more expensive than conventional PCR

-Requirement for 1000x less RNA than conventional assays

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Disadvantages

Assays require skill and competence

High equipment costs

Not ideal for multiplexingdepends on

machine

Lability of RNA

Contamination by DNA

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ReferencesMcPherson MJ, Mller SG (2006) PCR: The Basics.

BIOS, Oxford.

ISBN: 9780415355476JRUL: Blue, Floor 2: 572.808/MCP

Also electronic resource:

http://www.netLibrary.com/urlapi.asp?action=summary&v=1&bookid=53951

Kleppe K, Ohtsuka E, Kleppe R, Molineux I and Khorana HG (1971) Studies onpolynucleotides. XCVI. Repair replications of short synthetic DNAs as catalysed by DNApolymerase. J Mol Biol 56: 341-361.

Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA and Arnheim N (1985)Enzymatic amplification of -globin genomic sequences and restriction site analysis fordiagnosis of sickle-cell anaemia. Science 230: 1350-1354.

Saiki RK, Walsh PS, Levenson CH and Erlich HA (1989) Genetic analysis of amplified DNAwith immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA 86:6230-6234.

Mullis KB (1990) The unusual origin of the Polymerase Chain Reaction. Scientific American262: 56-61.

http://www.netlibrary.com/urlapi.asp?action=summary&v=1&bookid=53951http://www.netlibrary.com/urlapi.asp?action=summary&v=1&bookid=53951http://www.netlibrary.com/urlapi.asp?action=summary&v=1&bookid=53951http://www.netlibrary.com/urlapi.asp?action=summary&v=1&bookid=53951