Polymerase Chain Reaction (PCR


PCR Recipe
• Template DNA 50-100ng/μl • 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 μM each dNTP • Primers (0.1 and 0.5 μM) • DNA polymerase -1-2 unit/25 μl reaction

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

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 μl 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 μl of the supernatant into a new microfuge tube Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.

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., 95°C) 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.

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 two complementary stands is ideal. • PCR is carried out as usual, but with a great excess of one primers for the chosen strand.

Nested PCR
Two pairs (instead of one pair) of PCR primers are used to amplify a fragment. • First pair -amplify a fragment similar to a standard PCR. Second pair of primers-nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. • Advantage- Very low probability of nonspecific amplification

AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping individuals for a large number of loci •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.

Inverse PCR
• Inverse PCR (Ochman et al., 1988) uses standard PCR (polymerase chain reaction)- primers oriented in the reverse direction 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 ligated to generate circular DNA. Applications • Amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts.

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.

In Situ PCR
• In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can 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

In Situ PCR

Long PCR
• Extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb). • Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together. • Application- to clone large genes not possible with conventional PCR.

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.

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 is much 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 a sequence


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.

History of Real Time PCR
Initial work by Higuchi and first demonstrated the simultaneous amplification and detection of specific DNA sequences in realtime 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 device camera (Higuchi et al., 1993).


Real Time PCR
Introduction General Principles & Concepts

What are Fluorescent dyes?
Fluorescence Resonance Energy Transfer (FRET)

Some commonly used flurophores for labeling probes
Quantitating Fluorescence Improving Fluorescence Signal Detection

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 the sample. 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 of optical filters. • The wavelength (nm) where photon energy is most efficiently captured is defined as the Absorbancemax & the wavelength (nm) where light is most efficiently released is defined as the Emissionmax.

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 emitted fluorescent light (quantum efficiency). Environmental factors: Environmental conditions can affect the brightness or the wavelength of the absorption or emission peaks.

What is Fluorescence Resonance Energy Transfer (FRET)? 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 acceptor molecule without emission of a photon

FRET (cont’d):
The Donor and Acceptor in close physical proximity (10 -100 Angstrom) can lead to FRET or Quenching
hv hv






(a) Physical proximity + hv (FRET +ve)

(b) No physical proximity + hv

(c) No hv

Hybridization probes


(e) No Physical proximity + hv (Quenching released)

(d) Physical proximity + hv (Quenching)

TaqMan & Beacon Probes

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.

Real Time PCR Instruments


    

LightCycler (Idaho Technologies Roche)
Rotor-Gene (Corbett Research) iCycler (BioRad) Mx4000™ Multiplex Quantitative PCR System ABI Prism 7700 (Perkin-Elmer-Applied-Biosystem) SmartCycler (Cephid)

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

Real Time Detection

1a. Excitation filters 1b. Emission filters Tungsten halogen light source Microplate format
(350 - 1000nm continuous)


iCycler from BioRad

Probe types & Design

dsDNA BindingDye
      

SYBR Green I SYBR Green II EVA Green LC Green BEBO YO-PRO SYTO family

Sybr Green PCR Assay
Stronger signal Higher selectivity for dsDNA Lesser sequence dependent Higher stability Lesser inhibitory for Taq Higher resolution in melting curves

Less hazardous and mutagenicity)

Binds to Non specific PCR product Primer dimer

Hydrolysis Probes (TaqMan)

TaqMan Probe
When intact, the fluorescence of the reporter is quenched due to its proximity to the quencher 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
 Highly fluorogenic
 Easy PCR setup  Sequence-specific detection, multiplexing

 Expensive
 Probe design and positioning challenging  Similar conditions for primers and probes

 Elevated background (Quenching capacity)
 Probe degraded: no end-point analysis

Hairpin probes: Molecular beacons
Molecular Beacons are hairpin structures composed of a (25–40 nt) nucleotide base paired stem and a target specific nucleotide loop. The loop consists of target specific nucleotide (probe) sequences (15–30 nt) A fluorescent moiety (reporter)is attached to 5’ end and a quencher moiety is attached to 3’end. The stem keeps both the moieties in close proximity so that fluorescence is quenched.



5’ 3’

3’ 5’

5’ 3’ 5’




Operation of Molecular Beacon (MB): MB is non-fluorescent due to close proximity of the nonfluorescent quencher (Q) and the fluorescent 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 destroyed during amplification, because a DNA polymerase lacking 5' exonuclease activity is used


Primer molecular Beacon annealing

5’ 3’


5’ 3’ 5’

5’ 3’



Molecular beacons
 High specificity, low background
 Post PCR analysis  PCR multiplex  Allelic discrimination (greater specificity than linear probes)

 Challenging design
 Long probes – less yield  Intramolecular competitive binding  Low signal levels (proximity of reporter and quencher)

Scorpion Primers
Complementary sequence

5’ Reporter

3’ Quencher Blocker

PCR primer

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 probe hybridizes 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 The primer is part of the Scorpion probe

Scorpion stem-loop format
Primer, stopper to prevent read PCR through, probe sequence, fluorophore & quencher (detection system).
The primer is extended The primer binds to the target

The probe binds to the complimentary sequence of the DNA

Hybridization Probes
 These assays use two sequence-specific oligonucleotide probes in addition to two sequence specific primers. The two probes are designed to bind to adjacent sequences in the target. The probes are labeled with a pair of dyes that can engage in FRET. The donor dye is attached to the 3' end of the first probe, while the acceptor dye is attached to the 5' end of the second probe.  During real-time PCR, excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. At the annealing step, the probes hybridize to their target sequences in a head-to-tail arrangement. This brings the donor and acceptor dyes into proximity, allowing FRET to occur.  The increase in PCR product is proportional to amount of fluorescence

Hybridization probes
Probe 2

Probe 1

Probes hybridize to their target sequences in a head-to-tail arrangement.


Hybridization probes


Probe 2

Probe 1




LC red 640

Hybridization probes
– Probe with only one fluorophore – Easy synthesis and quality controls – Reduced background fluorescence – High specificity

• Strict compatibility between donor & acceptor fluorophores



Similar 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.


PolyA Tail

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
TaqMan probes

Molecular Beacons


FRET probes


LUX fluorogenic primers MGB Eclipse probes





Scorpion probes










Detection Chemistries

Real-Time PCR Terminology
•Amplification plot is the plot of fluorescence signal versus cycle number.

•Initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline of amplification plot. •An increase in fluorescence above the baseline indicates detection of accumulated PCR product.

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

Effect of Limiting Reagents

During the exponential phase, none of the reaction components is limiting; as a result, CT values are very reproducible for reactions with the same starting copy number. On the other hand, the amount of PCR product observed at the end of the reaction is very sensitive to slight variations in reaction components.

Threshold cycle CT
•The Threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. •The threshold line is set in the exponential phase of the amplification for the most accurate reading. •The cycle at which the sample reaches this level is called the Cycle Threshold, CT.

•CT value of 40 or more means no amplification and cannot be included in the calculations. •A sample whose Ct is 3 cycles earlier than another's has 23 = 8 times more template.

•Rn+ 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 of
the magnitude of the signal generated by the PCR

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

Standard Curve
A dilution series of known template concentrations can be used 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 that concentration (y-axis). From this standard curve, information about the performance of the reaction as well as various reaction parameters (slope, y-intercept,correlation coefficient).

Correlation coefficient (R2)
The correlation coefficient is a measure of accuracy of standard curve. Ideally, R2 = 1, although 0.999 is generally the maximum value.

A PCR efficiency of 100% corresponds to a slope of –3.32. 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 influence efficiency

Melt Curve Analysis
A melting curve charts the change in fluorescence observed when dsDNA with incorporated dye molecules “melts”, into ssDNA as the temperature of the reaction is raised. when double-stranded DNA bound with SYBR Green I dye is heated, a sudden decrease in fl uorescence is detected when the melting point (Tm) is reached. The fluorescence is plotted against temperature, and then the –ΔF/ΔT (change in fluorescence/change in temperature) is plotted against temperature to obtain a clear view of the melting dynamics.

Melt Curve Analysis

The probe-based technique is sensitive enough to detect SNP and can distinguish between homozygous wild type, heterozygous and homozygous mutant alleles by virtue of the dissociation patterns produced.

Melt Curve Analysis
Example: Presence of Primer Dimers

Absolute Quantification
•Requires the construction of an absolute standard curve for each target

•The standard curve is based on a serial dilution of a sample with known copy number
•Ct of each standard sample is plotted against the logarithm of the known concentration •The standard curve is then used to estimate concentrations of unknown samples

Standard Curve for Absolute quantification

Good efficiency, good sensitivity and good predictive power.

Relative Quantification
Housekeeping gene: Abundantly and constantly expressed gene. Expression level of these genes remains constant. eg 18 S rRNA, GAPDH, β Actin Normalization: To accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples.

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 are normalized 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)


Application in Molecular Diagnostics
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

Detection of Pathogens
A Scorpion Probe Based Real-Time PCR Assay for Detection of E. coli O157:H7 in Dairy Products.

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

Genomic DNA isolation
Primer-probes were designed based on eaeA gene sequences

standard curve preparation of 10fold serial dilution

Sensitive -2log CFU/mL
Singh et al.,2009

SNP Genotyping
Multiplex TaqMan assay for SNP genotyping

SNP Identification Molecular Beacons

High Resolution Melting
Mutations in PCR products are detectable by HRM analysis because they lead to changes in DNA melting curves. The A:T to G:C interchange, which is the most common SNP results in a difference of about 1 °C in Tm, which is readily detected by HRM. Dyes used for HRM analysis:

•SYTOR 9 dye (Invitrogen)
•LCGreenR, LCGreenR Plus+ (Idaho Technologies) •EvaGreen™ dye (Biotium Inc.) •SYBRR GreenER™ dye (Invitrogen)

DNA Methylation Analysis
Methylation influence gene expression by affecting the interactions with DNA of both chromatin proteins and specific transcription factors. Bisulfite treatment converts cytosine to uracil while 5-methy cytosine is resistant to the conversion.

Methylated DNA having “C” will have a higher melting temperature than unmethylated DNA having “T” at same position. This can be detected by melt curve analysis.

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