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Polymerase Chain Reaction

(PCR)

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PCR Recipe

Template DNA 50-100ng/l

Reaction buffer (Tris-HCl, ammonium ions, KCl),

magnesium ions, bovine serum albumin)

This buffer provides the ionic strength and

buffering capacity needed during the reaction.

MgCl2 - 1.5- 3mM

dNTPs -Equimolar ratios, 200 M each dNTP

Primers (0.1 and 0.5 M)

DNA polymerase -1-2 unit/25 l reaction

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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

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Colony PCR

Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct

ligation or plasmid products.

Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 l of TE

autoclaved dH2O in an microfuge tube.

Heat the mix in a boiling water bath (90-100C) for 2 minutes

Spin sample for 2 minutes high speed in centrifuge.

Transfer 20 l of the supernatant into a new microfuge tube

Take 1-2 l of the supernatant as template in a 25 l PCR standard PCR

reaction.

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Hot Start PCR This is a technique that reduces non-specific amplification during

the initial set up stages of the PCR

The technique may be performed manually by heating the reaction

components to the melting temperature (e.g., 95C) before adding

the polymerase

Specialized enzyme systems have been developed that inhibit the

polymerase's activity at ambient temperature, either by the binding

of an antibody or by the presence of covalently bound inhibitors

that only dissociate after a high-temperature activation step

DNA Polymerase- Eubacterial type I DNA polymerase, Pfu

These thermophilic DNA polymerases show a very small polymerase

activity at room temperature.

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Asymmetric PCR

Asymmetric PCR is used to preferentially amplify one

strand of the original DNA more than the other. It finds use in some types of sequencing and

hybridization probing where having only one of the twocomplementary stands is ideal.

PCR is carried out as usual, but with a great excess of oneprimers for the chosen strand.

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Nested PCR

Two pairs (instead of one pair) of PCR primers areused to amplify a fragment.

First pair -amplify a fragment similar to astandard PCR. Second pair of primers-nestedprimers (as they lie / are nested within the firstfragment) bind inside the first PCR productfragment to allow amplification of a second PCR

product which is shorter than the first one.

Advantage- Very low probability of nonspecificamplification

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AFLP PCR

Genomic DNA is digested with one or more

restriction enzymes. tetracutter (MseI) and a

hexacutter (EcoRI).

Ligation of linkers to all restriction fragments.

Pre-selective PCR is performed using primers

which match the linkers and restriction site specific

sequences.

Electrophoretic separation and amplicons on a gel

matrix, followed by visualisation of the band

pattern.

AFLP is a highly sensitive PCR-basedmethod for detecting polymorphisms in

DNA. AFLP can be also used forgenotyping individuals for a largenumber of loci

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Inverse PCR

Inverse PCR (Ochman et al., 1988) uses standard PCR(polymerase chain reaction)- primers oriented in the reversedirection of the usual orientation.

The template for the reverse primers is a restriction fragment

that has been selfligated Inverse PCR functions to clone sequences flanking a known

sequence. Flanking DNA sequences are digested and then ligatedto generate circular DNA.

Applications

Amplification and identification of sequences flankingtransposable elements, and the identification of genomic inserts.

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Multiplex PCR

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

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In Situ PCR

In Situ PCR (ISH) is a polymerase chain reaction that actuallytakes place inside the cell on a slide. In situ PCR amplificationcan be performed on fixed tissue or cells.

Applies the methodology of hybridization of the nucleic acids.

Allows identification of cellular markers

Limited to detection of non-genomic material such as RNA,genes or genomes

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In Situ PCR

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Long PCR

Extended or longer than standard PCR, meaning over 5kilobases (frequently over 10 kb).

Long PCR is useful only if it is accurate. Thus, special mixtures

of proficient polymerases along with accurate polymerasessuch as Pfu are often mixed together.

Application- to clone large genes not possible with

conventional PCR.

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Reverse Transcriptase PCR

Based on the process of reverse transcription, which reversetranscribes RNA into DNA and was initially isolated fromretroviruses.

First step of RT-PCR - "first strand reaction-Synthesis of cDNAusing oligo dT primers (37C) 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 sequencesby amplifying its complementary DNA.

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Allele-specific PCR

Used for identify of SNPs.

It requires prior knowledge of a DNA sequence,including differences between alleles.

Uses primers whose 3' ends encompass the SNP

PCR amplification under stringent conditions ismuch less efficient in the presence of a mismatch

between template and primer Successful amplification with an SNP-specific

primer signals presence of the specific SNP in asequence

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RealTime-PCR

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What is Real Time PCR?

Real Time PCR is a technique in which

fluoroprobes bind to specific target regions of

amplicons to produce fluorescence during PCR.

The fluorescence, measured in Real Time, is

detected in a PCR cycler with an inbuilt filter

flurometer.

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History of Real Time PCR

Initial work by Higuchi and first demonstrated the simultaneousamplification and detection of specific DNA sequences in real-

time by simply adding ethidium bromide (EtBr) to the PCR

reaction so that the accumulation of PCR product could be

visualised at each cycle. (Higuchi et al., 1992)

When EtBr is bound to double-stranded DNA and excited by

UV light it fluoresces.

Kinetic PCR: Continuously measuring the increase in EtBr

intensity during amplification with a charge-coupled devicecamera (Higuchi et al., 1993).

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Introduction

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What are Fluorescent dyes?

When a population of fluorochrome molecules is excited by light of an

appropriate wavelength, fluorescent light is emitted. The light intensity

can be measured by flurometer or a pixel-by-pixel digital image of thesample.

Excitation and Emission: Fluorodyes absorb light at one wavelength &

thereby boosts an electron to a higher energy shell.

The excited electron falls back to the ground state and the flurophore

re- emits light but at longer wavelength.

This shift makes it possible to separate excitation light from emission

light with the use ofoptical filters.

The wavelength (nm) where photon energy is most efficiently capturedis defined as the Absorbancemax & the wavelength (nm) where light is

most efficiently released is defined as the Emissionmax.

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What are Fluorescent dyes?

The range for which flurodyes absorb light is small (~ < 50nm)

and light outside this range will not cause the molecule to fluoresce.

Linearity: The intensity of the emitted fluorescent light is a linear

function of the amount of fluorochrome present. The signal

becomes nonlinear at very high fluorochrome concentrations.

Brightness: Fluorochrome differ in intensity.Dull fluorochrome is a less sensitive probe than a bright

fluorochrome. The brightness depends on two properties of the

fluorochrome-

Its ability to absorb light (extinction coefficient).

The efficiency with which it converts absorbed light into emittedfluorescent light (quantum efficiency).

Environmental factors: Environmental conditions can affect the

brightness or the wavelength of the absorption or emission peaks.

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FRET is a distance dependent interaction

between the excited states of 2 dye

molecules in which excitation is transferred

from a donor molecule to an acceptormolecule without emission of a photon

What is Fluorescence Resonance EnergyTransfer (FRET)?

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The Donor and Acceptor in close physical proximity

(10 -100 Angstrom) can lead to FRET or Quenching

hv

(b) No physical proximity +hv

D

(c) No hv

(d) Physical proximity +hv

R

hv

Q

(Quenching)

(e) No Physical proximity + hv

R

hv

Q

(Quenching released)

hv

(a) Physical proximity +hv

DA

(FRET +ve)Hybridization probes

TaqMan & Beacon Probes

AD

A

FRET (contd):

Q i i Fl

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Quantitating Fluorescence

A flurometer exploits the principles of fluorescence to quantitate

fluorescent (dye) molecules in the following way:

A strong light source which produces light within a specific light

range ( eg xenon arc lamp) is focused down to a tight beam.

The tight beam of light is sent through a filter which removes

most of the light outside of the target wavelength range.

The filtered light beam passes through the liquid target sample

striking some of the fluorescent molecules in the sample.

Light emitted from the fluorescent molecules travels orthogonal

to the excitation light beam pass through a secondary filter that

removes most of the light outside of the target wavelength range.

The filtered light then strikes a photodetector or

photomultiplier which allows the instrument to give a relative

measurement of the intensity of the emitted light.

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http://www.biosearchtech.com/download/brochures/bti_bhq_selectionchart.pdf

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Real Time PCR Instruments

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LightCycler (Idaho Technologies Roche)

Rotor-Gene (Corbett Research)

iCycler (BioRad)

Mx4000 Multiplex Quantitative PCR System

ABI Prism 7700 (Perkin-Elmer-Applied-Biosystem)

SmartCycler (Cephid)

Instruments

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General Description of Instruments

1. PCR cycler:1. 96 well format, 8 tube format, capillary (glass)

2. Air or block heater

3. Temperature ramp, temperature gradient

2. Fluorescence emission & detection :1. Fluorometer

2. CCD camera

3. Excitation source: xenon, halogen, laser

3. Fluorescent Dye Labeling of:

1. Oligonucleotides

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iCycler from BioRad

1a. Excitation filters1b. Emission filtersTungsten halogen light source

Cycler

Real Time Detection

Microplate format(350 - 1000nm continuous)

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Probe types & Design

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dsDNA BindingDye

SYBR Green I

SYBR Green II

EVA Green

LC Green

BEBO

YO-PRO

SYTO family

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Sybr Green PCR Assay

Stronger signal

Higher selectivity for dsDNA

Lesser sequence dependent

Higher stability

Lesser inhibitory forTaq

Higher resolution in melting curves

Less hazardous and mutagenicity)

Binds to

Non specific PCR product

Primer dimer

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Hydrolysis Probes (TaqMan)

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When intact, the fluorescence of the reporter

is quenched due to its proximity to thequencher

Probe hybridizes to the target

dsDNA-specific 5'>3' exonuclease activity

of Taq or Tth cleaves off the reporter

Reporter is separated from the quencher.

Fluorescent signal

Signal is proportional to the amount of

amplified product in the sample

TaqMan Probe

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Advantages

Highly fluorogenic

Easy PCR setup

Sequence-specific detection, multiplexing

Disadvantages

Expensive

Probe design and positioning challenging

Similar conditions for primers and probes

Elevated background (Quenching capacity)

Probe degraded: no end-point analysis

TaqMan Probe

H i i b M l l b

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Loop

Molecular Beacons are hairpin structures composed of a (2540 nt)nucleotide

base paired stem and a target specific nucleotide loop.

The loop consists of target specific nucleotide (probe) sequences (1530 nt)

A fluorescent moiety (reporter)is attached to 5 end and a quencher moiety is

attached to 3end. The stem keeps both the moieties in close proximity so that

fluorescence is quenched.

Stem

Hairpin probes: Molecular beacons

5 3

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Denaturation

Primer molecular

Beacon annealing

Extension

53Q

35

5

5

33

5

53

35

5

35

53

5

5QR

5

Operation of Molecular Beacon

(MB): MB is non-fluorescent due to

close proximity of the non-

fluorescent quencher (Q) and thefluorescent Reporter

The probe denatures and the loop

anneals to the target sequence of

the amplicon

Separating the quencher from the

fluorophore and thereby producing

fluorescence which is proportional to

the amplicons produced during PCR

MB is displaced not destroyedduring amplification, because a DNA

polymerase lacking 5' exonuclease

activity is used

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Molecular beacons

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Molecular beacons

Advantages

High specificity, low background

Post PCR analysis

PCR multiplex

Allelic discrimination (greater specificity than linear probes)

Disadvantages

Challenging design

Long probes less yield

Intramolecular competitive binding

Low signal levels (proximity of reporter and quencher)

Scorpion Primers

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Scorpion Primers

3 Quencher

Blocker

5 Reporter

Complementary sequence

PCRprimer

Scorpion primer consists of:

The loop of the Scorpions probe includes a sequence that is complementary to

an internal portion of the sequence it primes.

During the first amplification cycle, the Scorpions primer is extended, and the

sequence complementary to the loop sequence is generated.

After subsequent denaturation and annealing, the loop of the Scorpions probehybridizes to the internal target sequence, and the reporter is separated from the

quencher. The resulting fluorescent signal is proportional to the amount of

amplified product in the sample.

The Scorpions probe contains a PCR blocker just 3' of the quencher to prevent

read-through during the extension of the opposite strand.

The template &probe denature

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The primer ispart of theScorpion probe

The primer isextended

probe denature

The primer bindsto the target

Scorpion stem-loop

format

Primer, stopper to prevent

read PCR through, probe

sequence, fluorophore &

quencher (detection system).

The probe binds to thecomplimentary sequenceof the DNA

Hybridization Probes

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Hybridization Probes

These assays use two sequence-specific oligonucleotide probes in

addition to two sequence specific primers. The two probes aredesigned to bind to adjacent sequences in the target. The probes arelabeled with a pair of dyes that can engage in FRET. The donor dye isattached to the 3' end of the first probe, while the acceptor dye isattached to the 5' end of the second probe.

During real-time PCR, excitation is performed at a wavelength specificto the donor dye, and the reaction is monitored at the emissionwavelength of the acceptor dye. At the annealing step, the probeshybridize to their target sequences in a head-to-tail arrangement. Thisbrings the donor and acceptor dyes into proximity, allowing FRET to

occur.

The increase in PCR product is proportional to amount of fluorescence

H b idi ation p obes

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Hybridization probes

Probe 2

Probes hybridize to their

target sequences in a

head-to-tail arrangement. FRET

Probe 1

Hybridization probes

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Hybridization probes

h

D

A

h

AD

FRET

Amplicon

Probe 1

Probe 2

D FAM A LC red 640

Hybridization probes

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Hybridization probes

Advantages

Probe with only one fluorophore

Easy synthesis and quality controls

Reduced background fluorescence

High specificity

Disadvantages

Strict compatibility between donor & acceptor fluorophores

hAD

FRET

SUNRISE UNIPRIMER PROBE

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Q

PolyA Tail

SUNRISE UNIPRIMER PROBESimilar to Molecular Beacon except the stem contains a

poly A (15 mer) tail. This tail is complimenatry to the polyT

tail of one f the primers.

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QR

Q R

hv

AAAAAAAAAAAAAA

Primer with polyT tail

Sunrise Probe with polyA tail

binds to the primer polyT tail at

annealing.

The Sunrise probe changes conformation during

denaturation & quenching by DABCYL is removed

allowing FITC to fluoresce

Sunrise UniPrimer Probe is a modification of Molecular Beacon

Commonly Used Fluorescent Probes

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Commonly Used Fluorescent Probes

Detection Chemistries

2%

3%

9%

9%

15%

19%

78%

0% 10% 20% 30% 40% 50% 60% 70% 80%

TaqMan probes

Molecular Beacons

FRET probes

LUX fluorogenic

primers

MGB Eclipse probes

Other

Scorpion probes

R l Ti PCR T i l

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Real-Time PCR Terminology

Amplification plot is the plot of

fluorescence signal versus cyclenumber.

Initial cycles of PCR, there islittle change in fluorescencesignal. This defines the

baseline of amplification plot.

An increase in fluorescenceabove the baseline indicatesdetection of accumulated PCRproduct.

The parameter CT(Threshold cycle) is defined as thefractional cycle number at which the fluorescence passes thefixed threshold.

Effect of Limiting Reagents

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Effect of Limiting Reagents

During the exponential phase, none of the reaction components islimiting; as a result, CT values are very reproducible for reactionswith the same starting copy number.

On the other hand, the amount of PCR product observed at the end ofthe reaction is very sensitive to slight variations in reaction components.

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The Threshold line is the level

of detection or the point at whicha reaction reaches a fluorescent

intensity above background.

The threshold line is set in theexponential phase of the

amplification for the mostaccurate reading.

The cycle at which the samplereaches this level is called theCycle Threshold, C

T.

Threshold cycle CT

CT value of 40 or more means no amplification and cannotbe included in the calculations.

A sample whose Ct is 3 cycles earlier than another's has23 = 8 times more template.

Rn

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DRnRn+is the Rn value of a reaction containing all components (the

sample of interest)

Rn- is the Rn value detected in NTC (baseline value)

DRn is the difference between Rn+ and Rn-. It is an indicator ofthe magnitude of the signal generated by the PCR

DRn is plotted against cycle numbers to produce theamplification curves and to estimate the CT values

St d d C

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A dilution series of known template concentrations can beused to establish a standard curve for determining the

initial starting amount of the target template.

The log of each known concentration in the dilution series(x-axis) is plotted against the Ct value for thatconcentration (y-axis).

From this standard curve, information about theperformance of the reaction as well as various reactionparameters (slope, y-intercept,correlation coefficient).

Standard Curve

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Correlation coefficient (R2)

The correlation coefficient is a measure of accuracy of

standard curve. Ideally, R2 = 1, although 0.999 is generallythe maximum value.

Efficiency

A PCR efficiency of 100% corresponds to a slope of3.32.

Ideally, the efficiency (E) of a PCR reaction should be 100%but experimental factors such as the length, secondary

structure, and GC content of the amplicon can influenceefficiency

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Melt Curve Analysis

A melting curve charts the change in fluorescenceobserved when dsDNA with incorporated dye moleculesmelts, into ssDNA as the temperature of the reaction israised.

when double-stranded DNA bound with SYBR Green I dyeis heated, a sudden decrease in fl uorescence is detectedwhen the melting point (Tm) is reached.

The fluorescence is plotted against temperature, and then

the F/T (change in fluorescence/change intemperature) is plotted against temperature to obtain aclear view of the melting dynamics.

Melt Curve Analysis

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Melt Curve Analysis

The probe-based technique is sensitive enough to detect SNP and candistinguish between homozygous wild type, heterozygous andhomozygous mutant alleles by virtue of the dissociation patternsproduced.

Melt Curve Analysis

http://en.wikipedia.org/wiki/Homozygoushttp://en.wikipedia.org/wiki/Wildtypehttp://en.wikipedia.org/wiki/Heterozygoushttp://en.wikipedia.org/wiki/Mutanthttp://en.wikipedia.org/wiki/Mutanthttp://en.wikipedia.org/wiki/Heterozygoushttp://en.wikipedia.org/wiki/Wildtypehttp://en.wikipedia.org/wiki/Wildtypehttp://en.wikipedia.org/wiki/Wildtypehttp://en.wikipedia.org/wiki/Homozygous

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Example: Presence of Primer Dimers

Melt Curve Analysis

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Requires the construction of an absolute standard curve foreach target

The standard curve is based on a serial dilution of a sample

with known copy numberCt of each standard sample is plotted against the logarithmof the known concentration

The standard curve is then used to estimate concentrations

of unknown samples

Absolute Quantification

Standard Curve for Absolute quantification

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Standard Curve for Absolute quantification

Good efficiency,good sensitivity and

good predictivepower.

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Relative Quantification

Housekeeping gene: Abundantly and constantlyexpressed gene. Expression level of these genes remainsconstant. eg 18 S rRNA, GAPDH, Actin

Normalization: To accurately quantify gene expression,the measured amount of RNA from the gene of interest isdivided by the amount of RNA from a housekeeping genemeasured in the same sample to normalize for possible

variation in the amount and quality of RNA betweendifferent samples.

C ti C M th d

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Comparative Ct Method.

This involves comparing Ct values of the samples with a

control or calibrator such as a non-treated sample.

The Ct values of both the calibrator and the samples arenormalized to an endogenous housekeeping gene.

This give Ct value of control and the sample.The comparative Ct method is also known as 2

-Ct method,where Ct = Ct,sample - Ct,reference

Fold change = Efficiency-Ct or 2-Ct

(which gives relative gene expression)

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REALTIME-PCRAPPLICATIONS

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Clinical microbiology and Food microbiology

Gene expression

viral quantitation

Single Nucleotide Polymorphism (SNP) analysis

Clinical oncology

Cancer

Analysis of cellular immune response in peripheral blood

Chromosome aberrations

Application in Molecular Diagnostics

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Detection of PathogensA Scorpion Probe Based Real-Time PCR Assay for Detection of E. coliO157:H7 in Dairy Products.

Singh et al.,2009

RTi-PCR method based on Scorpion probe targeting theeae gene of E. coli O157:H7.

Genomic DNA isolation

Primer-probes were designedbased on eaeA gene sequences

standard curve preparation of 10-

fold serial dilution

Sensitive -2log CFU/mL

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SNP Genotyping

Multiplex TaqMan assay for SNP genotyping

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SNP Identification Molecular Beacons

High Resolution Melting

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High Resolution Melting

Mutations in PCR products are detectable by HRM analysisbecause they lead to changes in DNA melting curves.

The A:T to G:C interchange, which is the most common SNPresults in a difference of about 1 C in Tm, which is readilydetected by HRM.

Dyes used for HRM analysis:

SYTOR 9 dye (Invitrogen)

LCGreenR, LCGreenRPlus+ (Idaho Technologies)EvaGreen dye (Biotium Inc.)

SYBRR GreenER dye (Invitrogen)

DNA Methylation Analysis

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DNA Methylation Analysis

Methylation influence gene expression by affecting the

interactions with DNA of both

chromatin proteins andspecific transcription factors.

Bisulfite treatment converts cytosine to uracil while5-methy cytosine is resistant to the conversion.

Methylated DNA having C will have a higher meltingtemperature than unmethylated DNA having T at sameposition.

This can be detected bymelt curve analysis.