basic real time pcr technique_jan 2015

8/9/2019 Basic Real Time PCR Technique_Jan 2015

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Sawitree Suklour (Meow)


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PCR ProcessPCR Process

1. Temperature Cycling

Denaturation 94Annealing 55

2. Every cycle DNAbetween primers is

duplicated


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PCR Amplification


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Exponential AmplificationExponential Amplification

30 cycles --- 1 billion copies in theory


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WhatWhat is RTis RT--PCRPCR??

RT-PCR: Reverse Transcription Polymerase

Chain ReactionA method to amplify a piece of a ribonucleic acid (RNA)molecule. The RNA strand is first reverse transcribed into

s comp emen or comp emen ary o owe yamplification of the resulting DNA using PCR. This can

either be a 1 or 2 step process.


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One step Vs Two stepOne step Vs Two stepRTRT--PCRPCR

One stepcDNA synthesis to PCRamplification is performed in

a single tube.Minimizes experimental

variation because bothenzymatic reactions occur in

Uses RNA starting template.Prone to rapid degradation ifnot handled well.Not suitable in situations

where the same sample is

assayed on several occasionsover a period of time.Less sensitive than two stepprotocols


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One step Vs Two stepOne step Vs Two stepRTRT--PCRPCR

Two step

Reverse transcription and

PCR occur in separate tubesAllows several different realtime PCR assays on dilutionsof a single cDNA.

assays have the same amountof template to those assayedearlier.

Date from two step is quite

reproducible.Allow for increased DNAcontamination.


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RealReal--time PCRtime PCR

Real time PCR: a technique used to

monitor the progress of a PCRreaction in real time

Real Time PCR is based on thedetection of the fluorescenceproduced by a reporter moleculewhich increases


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Fluorescent reporter moleculesFluorescent reporter molecules

Dyes that bind to the double-stranded

DNA-SYBR Green

Sequence specific probes

- Hydrolysis probe

- Hybridization probe


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Hydrolysis probeHydrolysis probe1. TaqMan probe

The reporter dye (R) represents the quenching dye that disrupts

the observable signal from the quencher dye (Q)when it is within

a short distance.


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22. Molecular. Molecular BeaconsBeaconsMolecular beacons are hairpin

shaped molecules with aninternally quenched fluorophorewhose fluorescence is restored

Molecular Beacon Example Sequence

Fluorophore at 5' end;5GCGAGCTAGGAAACACCAAAGATGATATTTGCTCGC -3'-DABCYL

nucleic acid


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The oligonucleotide that consists of a probeand primer linked together is called a Scorpion

probe or Scorpion primer.

3. Scorpion probe

this technique isthat the detectionof the amplicon is

much faster dueto the unimolecularreaction.


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Hybridization probeHybridization probe


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Which RealWhich Real--Time Chemistry Is Right for You?Time Chemistry Is Right for You?


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What happens duringa PCR reaction ?


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0

200000000

400000000

600000000

800000000

1E+09

1.2E+09

1.4E+09

1.6E+09

0 5 10 15 20 25 30 35

AMOUNTOFDNA

PCR CYCLE NUMBER

CYCLE NUMBER AMOUNT OF DNA

0 1

1 2

2 4

3 84 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

1

10

10 0

1000

10000

100000

1000000

10000000

100000000

1000000000

10000000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AMOUNTO

FDNA

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 1,500,000,000

33 1,550,000,000 34 1,580,000,000


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Amplification curveAmplification curve


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Amplification curveAmplification curve

Baseline: The initial cycles of PCR, in which there is littlechange in fluorescence signal.

Threshold: The location on the graph where the values of thesamples are determined. The threshold should be set inthe region associated with an exponential growth of PCRpro uct.

CT: (threshold cycle) The cycle number at which thefluorescence passes the threshold. Lower Ct indicates

higher starting amount; lower Ct for the same startingamount indicates higher detection sensitivity


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Threshold lineThreshold line

The Threshold line is the level of detection or thepoint at which a reaction reaches a fluorescentintensity above background.

threshold

Ct


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Standard curve: a dilution series of known concentrations, used tocalculate unknown amount

Slope: indicates amplification efficiency, ideal slope = -3.32 with 100%efficiency, values between 90% -110% considered good

Correlation Coefficient (R2): indicates reproducibility, ideal R= 1.000, .

Y-intercetp: the y-intercept corresponds to the theoretical limit ofdetection of the reaction, or the Ct value expected if the lowest copynumber of target molecules denoted on the x-axis gave rise to

statistically significant amplification.


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Melting curve (disassociation curve)Melting curve (below, left): plot of fluorescence vs. temperature; donefollowing PCR cyclingDerivative (below, right):most common view; plot of dF/dT vs.temperature

Application: indicates specificity, ideal is single peak; multiple peaksindicate non-specific amplification including primer-dimers No-template control reactions must be run to ensure specificity


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Quantitative & Qualitative assay

in Real time PCR


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Absolute vs Relative Quantification


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Absolute quantification without

standard curve


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Absolute Quantification Using the

Standard Curve Method

The standard curve method for absolute quantification is similar to the

standard curve method for relative quantification, except the absolutequantities of the standards must first be known by some independent means.


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http://cels.uri.edu/gsc/cndna.html


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How to create the standard curve

- known copy number

or concentration.

- at least 3 points and

cover concentration

.

- duplicate or triplicate

of each point.

C id ti f d t d d


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Consideration of good standard

curve

l Q f


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Relative QuantificationMethods for relative quantitation of gene expression allowyou to quantify differences in the expression level of aspecific target (gene) between different samples. The data

output is expressed as a fold-change or a fold-difference ofexpression levels

- Comparative Ct method- delta delta Ct

Relative Quantitation


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Relative QuantitationComparative Ct (Ct Method)

Compares the Ct difference for calibrator (GOI minus

HK) and the sample (GOI minus HK)


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The Reaction Components

1) Target DNA, RNA - contains the sequence to be amplifie

2) Pair of Primers - oligonucleotides that definethe sequence to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building

blocks.

4) Taq polymerase, reverse transcriptase - enzyme thatcatalyzes the reaction

5) Mg++ ions - cofactor of the enzyme

6) Buffer solution maintains pH and ionic strength

of the reaction solution suitable for the activity of theenzyme

7) Fluorescent dye


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Commercial master mixes, which arewidely available,simplify theo timisation and are convenient.

PCR Master MixPCR Master Mix


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What is difference bet een SYBRWhat is difference bet een SYBR


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What is difference between SYBRWhat is difference between SYBR

green& SYBR GreenERgreen& SYBR GreenER


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Probe detection base

qPCR

- Platinum qPCR SuperMix-UDG

- Express qPCR SuperMix-UDG

Real time PCR Master MixReal time PCR Master Mix

- nnu x q as er x ro e

qRT-PCR-SuperScript III Platinum one-step qRT-PCR SuperMix

- Express one-step qRT-PCR SuperMix- InnuScript one-step qRT-PCR superMix

Ad t f th kitAd t f th kit


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Advantages of the kitsAdvantages of the kits


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Thank you for your attentionThank you for your attention

basic real time pcr technique_jan 2015

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