Chain Reaction (PCR)

• PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.

• As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in different samples. Polymerase Chain Reaction (PCR)

• PCR carry out for – DNA cloning – sequencing, – DNA-based phylogeny, or – functional analysis of genes; – the diagnosis of diseases; – genetic fingerprints and – the detection and diagnosis of infectious diseases. PROCEDURE ….. PCR Reagents

• Template DNA (e.g., DNA, genomic DNA). • Forward and reverse PCR primers. • MgCl2 (25 mM). • dNTPs (a mixture of 2.5 mM dATP, dCTP, dGTP, and dTTP). • PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 25°C. PCR Reagents

• Thermal stable DNA polymerase – DNA without 3′ → 5′ activity • Taq DNA polymerase • Tth DNA polymerase • Tfl DNA polymerase – DNA polymerases with 3′ → 5′ proofreading activity: • Pwo DNA polymerase • Pfu DNA polymerase • Tli DNA polymerase • Vent DNA polymerase PCR Reagents

• Optional: PCR additives /cosolvents (optional; e.g., • betaine, glycerol, DMSO, formamide, bovine serum albumin, ammonium sulfate, polyethylene glycol, gelatin, Tween-20, Triton X-100, β-mercaptoethanol, or tetramethylammonium chloride).

Polymerase Chain Reaction (PCR) • Initialization step: 94–96 °C (or 98 °C if extremely thermostable polymerases are used), for 1–9 minutes. – the complete separation of the DNA strands and unfolding secondary structures – This stage also required for DNA polymerases that require heat activation by hot-start PCR.

Polymerase Chain Reaction (PCR) • Denaturation step: 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. – it is appropriate to use a higher denaturation temperature and a longer incubation time for some templates, such as those templates with high GC content, to achieve complete denaturation for more complete denaturation of the DNA template • Although a higher temperature and a longer incubation time reduce lifetime of .

Polymerase Chain Reaction (PCR)

• Annealing step: 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3–5 °C below the Tm of the primers used. • Extension/elongation step (72 °C): The temperature at this step depends on the DNA polymerase used; – has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme. Polymerase Chain Reaction (PCR)

• In elongation step, – the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, – banding the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand (Phosphodiester band). – Primer extension time depends on the length and concentration of the target sequence, as well as the extension temperature. Extending the Chain DNA elongation DNA elongation DNA polymerases synthesize DNA using 5’dNTP substrates, a DNA template, and elongate the chain at the 3’ end:

Base1 Base 2 Base1 Base2 + OH PPP 3’ OH PPP OH PPP OPO 5’

B1 B2 B3 B4 B5 B6 B7 …

etc… PPP OH Polymerase Chain Reaction (PCR) • In elongation step, – Taq DNA polymerase extends at a rate of 0.25 per second at 22°C, 1.5 nucleotides per second at 37°C, 24 nucleotides per second at 55°C, greater than 60 nucleotides per second at 70°C, and 150 nucleotides per second at 75 to 80°C. – at 72°C, Taq DNA polymerase is expected to extend at the rate of greater than 3500 nucleotides per minute. – Thus, in common, an extension time of 1 min per kilobase is more than sufficient to generate the expected PCR product.

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)

• Final elongation: – This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended • (A tail for cloning). Polymerase Chain Reaction (PCR) • Two-step cycling programs are generally applied when a high annealing temperature is used, such as 65 to 70°C.

• Because a higher annealing temperature improves amplification specificity, it is argued by some investigators that better PCR results may be obtained using a two-step cycling program . Polymerase Chain Reaction (PCR)

• In long PCR, – Two-step cycling programs are more frequently used than three-step cycling programs. – For example, denaturation at 92 to 95°C for 10 to 30 s, followed by

annealing and extension at 65 to 68°C for 1 min per kilobase will increase the probability of obtaining the desired product.

Melting PCR 100 94 oC

50 Temperature

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Melting PCR 100 94 oC

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5’ 3’

Melting PCR 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C

50 50 oC Temperature

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5’ 5’ 3’ Temperature control in a PCR thermocycler

Temperature 0C

94 0C - denaturation

50 – 70 0C - primer annealing

72 0C - primer extension

94 0C - denaturation

Melting PCR 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C

50 50 oC Temperature

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Melting PCR 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C

50 50 oC Temperature

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Melting PCR 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C

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Melting PCR 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C

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Melting 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C

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3’ 5’ 5’ T i m e 5’ 5’ 5’ 5’ 3’

5’ Fragments of 5’ defined length 5’ 5’

5’ 5’ PCR Round 1

target DNA 5' 3' Double-stranded DNA 3' 5'

5' 3' Denaturation 3' 5'

5' 3' Primer annealing 3' 5'

5' 5' 3' 3' 5' 5' 3' 3' Extension 5' 5' 3' 3' 3' 3' 5' 5'

repeat PCR cycles DNA polymerase always adds nucleotides to the 3’ end of the primer 5' 3' 5' 3' PCR Round 2 5' 3' 3' 5'

After the second round of 5' 3' PCR, the number of long 3' 5' denaturation strands increases 5' 3' arithmetically and the 3' 5' number of short strands increases exponentially 5' 3' (the number of chromosomal strands is 3' 5' primer annealing 3' always the same). 5'

3' 5'

5' 3' 5' 3' 3' 5' 5' 3' extension Short strand 3' 5' 5' 3' 3' 5'

Long strand Chromosomal strand • The confirmation of PCR products 1. Correct product size (expected-size product) 2. Sequencing the products. 3. Use a gene probe to confirm the product.s 4. Use nested PCR PCR

• PCR amplifications can be grouped into three different categories: – standard PCR, – long PCR, and – multiplex PCR. • Standard PCR – amplification of a single DNA sequence that is less than 5 kb (my experience, 2kb) in length and – applications, such as sequencing, cloning, mutation detection, etc. PCR Optimisation

• Long PCR is used for – the amplification of a single sequence that is longer than 5 kb (2kb) and up to 40 kb in length. – Its applications include • long-range sequencing; • amplification of complete genes; • molecular cloning; • assembly and production of larger recombinant constructions for PCR-based mutagenesis. Long PCR

• Programming an increase in extension time automatically in later cycles may also improve the yields of the amplification. – Forexample: 3M*10s for extension time

• increasing the extension time in each of the later PCR cycles could increase the likelihood of synthesizing long PCR products. PCR Optimisation

• Multiplex PCR, – is used for the amplification of multiple sequences that are less than 5 kb in length. – Its applications include • pathogen identification (detecting in quarantine); • template quantitation; • genetic disease diagnosis; • population genetics. Multiplex-PCR • targeting multiple genes at once with multiple primer sets within a single PCR mixture which produce amplicons of varying sizes that are specific to different DNA sequences.

• Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction (very close Tm)

• amplicon length should be different enough to form distinct bands when visualized by gel electrophoresis. Multiplex PCR

• Use of multiple sets of primers • to detect more than one organism or • to detect multiple genes in one organism.

E. Coli Salmonella sp. genome genome

or Multiplex PCR

• Therefore, Multiplex PCR enabling simultaneous amplification of some genes in one reaction by using more than one pair of primers.

PCR Optimization • Optimization of PCR depend on: – (1) Quality and concentration of DNA template; – (2) Design and concentration of primers; – (3) Concentration of ; – (4) Concentration of the four deoxynucleotides (dNTPs); – (5) PCR buffer systems; – (6) Selection and concentration of DNA polymerase; – (7) PCR thermal cycling conditions; – (8) Addition and concentrations of PCR additives (enhancements) – (9) Use of the “hot start” technique. PCR Optimization- DNA template

• The quality and concentration of DNA templates can directly affect the outcome of PCR amplifications. – For amplification from genomic DNA, use 100 to 500 ng of template DNA (50µ). – for plasmid DNA very low concentration – In multiplex PCR, two- to five fold more DNA template than what is needed for a typical PCR should be used. PCR Optimization: Buffers • Taq DNA polymerase are performed in – 10 mM Tris- HCl – 50 mM KCl. • KCl facilitates primer binding but concentrations higher than 50mM inhibit Taq

• For Tth and Tfl DNA polymerases, and DNA polymerases with proofreading activity (Pwo, Pfu, Tli, and Vent DNA polymerases, – a buffer system of 50 mM Tris-HCl and 20 mM (NH4)2SO4 is normally used. – Concentrations of these can be altered PCR Optimization: Buffers • In long PCR, in addition to 25 mM Tris-HCl (pH 8.9 at 25°C) and 100 mM KCl – requires 20 to 25 mM Tricine, 85 mM potassium acetate, – DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet P-40, Triton X-100 can be added to aid in the PCR reaction • Enhance specificity, but also can be inhibitory – Pre-mixed buffers are available PCR Optimization: MgCl2

• MgCl2 affects – DNA polymerase activity

– DNA strand denaturation temperatures of both template and PCR product,

– primer annealing,

– PCR specificity,

– primer-dimer formation. PCR Optimization: MgCl2 • Excess magnesium gives non-specific binding result to accumulation of nonspecific amplified products seen as multiple bands on an agarose gel,

• Too little magnesium gives reduced yield

• concentration of MgCl2 should be higher than dNTP PCR Optimization: dNTPs • Concentration of dNTPs can affect the yield and specificity of a PCR amplification.

• Lower concentrations of dNTPs minimize mis-priming and reduce the likelihood of extending mis-incorporated nucleotides, which in turn increase specificity and fidelity of PCR amplifications.

• dNTPs concentration should determine based on

– the lowest dNTPs concentration result to maximum production

– Also, the length and composition of the target sequence. PCR Optimization: Primer Design

• Optimal Length 18-28 nucleotides

• Tm: For primers shorter than 20 bases, an estimate of Tm can be calculated as Tm = 4 (G + C) + 2 (A + T).

• Both of Primers should have melting temperatures that are within 2 to 5°C of each other. • • Primers with Tm higher than 50°C will generally provide specific and efficient amplifications. – For long PCR, a Tm of 62 to 70°C is recommended. PCR Optimization: Primer Design

• Annealing temperature 50oC-70oC – Annealing temperatures from 55 to 70°C generally yield the best results. • Preferably 58oC-63oC • A typical primer annealing temperature is 5°C below the calculated Tm of the primers. • Increasing the annealing temperature reduce incorrectly annealed primers and reduces mis-extension of incorrect nucleotides at the 3′ end of primers. Therefore, a higher annealing temperature increases amplification specificity. • Increase in annealing temperature result in reduced yield PCR Optimization: Primer Design

• GC content 40-60% increases specificity (higher Tm). • G or C at 3’ terminus – 2-3 GC in 5 last of 3 end – what is sticky end? • Avoid complementary sequences within a primer or between the two primers. – Complementary sequences in two primer increase formation of primer-dimers (hetero dimer) – Inner self complementarity (in each primer) increase secondary structures (Hairpins (loop) and self-dimer) • avoid continuous stretches of purines or pyrimidine, as well as multiple repeats of thymidine residues at the 3′ end of the primer.

PCR Optimization: DNA polymerase • Extension rate, processivity, fidelity (error rate), , and thermal activity profile of , are important in PCR. – Processivity: • the number of nucleotides replicated before the enzyme dissociates from the DNA template. – extension rate • Taq DNA polymerase has an extension rate of 35 to 100 nucleotides per second at 72°C. – Thermostability and thermal activity • The half-life of Taq DNA polymerase activity is more than 2 h at 92.5°C, 40 min at 95°C, and 5 min at 97.5°C which is sufficient to remain active over 30 or more cycles. PCR Optimization: DNA polymerase • Fidelity: show with error rate – The error rate for Taq DNA polymerase, which lacks proofreading 3′ → 5′ activity, is estimated at approx 1 to 2 × 10–5 errors per nucleotide per duplication.

– The estimated error rates in presence proofreading enzymes is approx 1 to 2 × 10–6 errors per nucleotide per duplication, • a 10-fold improvement over standard Taq DNA polymerase.

– proofreading enzymes with lower error rates also have lower extension rates, resulting in lower PCR efficiency. Therefore, more amplification cycles are required to achieve adequate amount of amplified DNA. PCR Optimization: additives or cosolvents

• PCR which use templates with high guanine/cytosine content or stable secondary structures, that may still amplify inefficiently, resulting in little or no desired product and/or nonspecific products. • • the most effective and frequently used strategy is addition of various organic additives or cosolvents. – dimethyl sulfoxide, Glycerol, Formamide, bovine serum albumin, (NH4)2SO4;, polyethylene glycol, gelatin, non-ionic detergents (such as Tween 20 and Triton X-100), β- mercaptoethanol, tetramethylammonium chloride, and N,N,N-trimethyglycine (betaine)

PCR Optimization: additives or cosolvents

• dimethyl sulfoxide (DMSO), formamide, glycerol, and polyethylene glycol, may affect the Tm of the primers, the thermal activity profile of Taq DNA polymerase and the degree of product strand separation. • Gelatin, bovine serum albumin, and nonionic detergents, such as Tween-20 and Triton X-100, are thought to stabilize DNA polymerases . • tetramethylammonium chloride (TMAC) is used to eliminate nonspecific priming. PCR Optimization: additives or cosolvents

• (NH4)2SO4 may increase the ionic strength of the reaction mixture, altering the denaturation and annealing temperatures of DNA, and may affect polymerase activity.

• Betaine increase the thermo-stability of DNA polymerases, as well as to alter DNA stability such that GC-rich regions melt at temperatures more similar to AT-rich regions. PCR Optimization: thermal cycling

• Optimization of PCR thermal cycling conditions includes – determination of cycle number, – the temperature and incubation time period for: • template denaturation, primer annealing, and primer extension. • The following will cause the reaction to terminate. – thermal inactivation of the DNA polymerase after each denaturation step, – reduction in denaturation efficiency, – the reduced efficiency of primer annealing After 25 cycles have 3.4 x 107 times more DNA 1. Exponential amplification 2. Leveling off (reductional) stage 3. Plateau

plateau is reached after 25-30 cycles

# PCR cycles PCR Optimization: thermal cycling • PCR Stages – Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present. • continue up until the point when the product reaches about 1012 molecule in a 100-μL reaction. – Leveling off (reductional) stage : The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting. – Plateau: No more products accumulate due to exhaustion of reagents and enzyme. (after about 20 to 40 cycles.) PCR Optimization: Cycle Number

• Half-life of Taq is 30 minutes at 95oC • Therefore if you use more than 30 cycles at denaturation times of 1 minute, the Taq will not be very efficient at this point

Theoretical yield = 2n ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc eg. if you start with 100 copies after 30 cycles you will have 107, 374, 182, 400 copies More Cycles = More DNA Size Number of cycles Marker 0 10 15 20 25 30 In summary

• Primer length should not exceed 30 mer.

• GC Content should be in the range of 40-60 % for optimum PCR efficiency. • • Primers should end (3′) in a G or C, or CG or GC: this increases efficiency of priming.

Variations of the PCR Touchdown PCR (Step-down PCR) • A variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses.

• The annealing temperature at the initial cycles is usually a

few degrees (3-5 °C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5 °C) below the

primer Tm.

• The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles. Helicase-dependent amplification

• Similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.

Reverse Transcription PCR (RT-PCR):

• For amplifying DNA from RNA. reverse transcribes RNA into cDNA, which is then amplified by PCR. – Reverse transcriptase was initially isolated from retroviruses.

• RT-PCR is widely used in expression profiling, – to determine the expression of a gene or • Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA. – to identify the sequence of an RNA transcript, – To determine transcription start and termination sites.

• If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. Reverse Transcriptase PCR

• Based on the process of reverse transcription:

– 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.

RT-PCR The enzyme reverse transcriptase is used to make a DNA copy (cDNA) of an RNA template from a virus or from mRNA.

Viral RNA BacterialmRNA Protozoan (eukaryotic) poly A mRNA

AAAA3’

Reverse transcriptase RNA Primer 5’ 5’ 3’ Extension

5’ RNA/cDNA 3’

3’ 5’ RNA Normal PCR with two primers 3’ cDNA 5’ Summary of RNA processing

• In eukaryotes, RNA polymerase produces a “primary transcript”, an exact RNA copy of the gene. • A cap is put on the 5’ end. • The RNA is terminated and poly-A is added to the 3’ end. • All introns are spliced out. • At this point, the RNA can be called messenger RNA. It is then transported out of the nucleus into the cytoplasm, where it is translated. Post Transcriptional Processing

• Primary transcripts of mRNA are called as heteronuclear RNA or heterogeneous nuclear RNA (hnRNA). • hnRNA are larger than matured mRNA by many folds. • Modification includes – Capping at the 5- end – Tailing at the 3- end – mRNA splicing Hot Start PCR • The “hot start” technique enhances PCR specificity by reducing of non-specific amplification and primer-dimers during the initial steps of PCR.

• DNA polymerases show a very small polymerase activity at room temperature and a brief incubation of a PCR mix at temperatures significantly below the Tm can result in primer-dimer formation and nonspecific priming.

• the Taq DNA polymerase is active at room temperature and to a lesser degree, even on ice.

Hot Start PCR

• The purpose of a hot start is to withhold one of the critical components from the reaction until the temperature in the first cycle rises above the annealing temperature.

• There are various methods of performing a hot start. – Manual hot start is performed by withholding one of the reaction components, such as the DNA polymerase or magnesium, and adding it only after the reaction temperature rises above 80°C during the first denaturation step. Hot Start PCR

• Wax-mediated hot start involves addition of a wax layer separating the component being withheld from the remainder of the reaction mix. During the temperature increase in the first denaturation step, the wax melts and the withheld component is mixed with the rest of the reaction components, starting the amplification reaction.

• Specialized enzyme systems have been developed that inhibit the polymerase's activity at low temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot Start PCR

• Hot start Taq DNA polymerase is constructed through the addition of an anti-Taq DNA polymerase antibody.

• The antibody will prevent the DNA polymerase activity until the temperature rises during the initial denaturation step. The increased temperature dissociates and degrades the bound antibody, initiating PCR amplification.

• Hot start is commonly used for multiplex, long PCR and DDRT-PCR amplifications.

Hot Start PCR

• Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient (environment) temperature and are instantly activated at elongation temperature.

• In my experience, I preheat (94℃) the thermal cycler before placing the tubes from ice into the thermal cycler. (Gene fishing in DDRT- PCR) SSR and ISSR- PCR

• A PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths. Nested PCR

• increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive (continious) PCRs.

• In the first reaction, one pair of primers is used to generate DNA products, which may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR

• Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences. 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.

• single- and double-tube nested PCR

• Advantage- Very low probability of nonspecific amplification

Seminested PCR

Three primers are required, the normal upstream and downstream primers as well as a third, internal primer. Two rounds of PCR are performed, a normal PCR with the upstream and downstream primer, and then a second round of PCR with the downstream and internal primer. A second smaller product is the result of the second round of PCR.

Downstream primer Internal primer

Upstream primer Normal 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 or 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.

Colony PCR in our laboratory

• The screening of bacterial (E. Coli) clones for correct ligation: – Pick a bacterial colony with an autoclaved yellow (100µL) tips, Mix (suspension) and shake it into 20 μl of autoclaved dH2O in an microfuge tube and make a bacterial suspension of E. coli. – Take 2 μl of the bacterial suspension as template in a 25 μl PCR standard PCR reaction. – store tube containing remains of bacterial suspension in refrigerator – if the clones is confirmed, culture bacterial suspension in LB media for Plasmid extraction and long-time conservation in -80C. Quantitative PCR (qPCR):

• It is also sometimes abbreviated to RT-PCR (real-time PCR) but this abbreviation should be used only for reverse transcription PCR. qPCR is the appropriate contractions for quantitative PCR (real-time PCR).

• Used to measure the quantity of a target sequence (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA.

Why real time PCR ?

• QUANTITATION OF mRNA – northern blotting – protection assay – in situ hybridization – RT-PCR • most sensitive • can discriminate closely related mRNAs • technically simple Quantitative PCR (qPCR):

• quantitative PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative PCR has a very high degree of precision.

Quantitative PCR methods use – fluorescent dyes, such as Sybr Green, EvaGreen or – fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time..

Real-Time PCR SYBR Green I

SYBR Green I is a fluorescent dye that upon intercalation into double stranded DNA exhibits an hn increase in fluorescence intensity of greater than 100-fold. Advantages include the relatively low hn cost of the dye, the fact that it will work with any primer set since it is not sequence specific, and the ability to perform a melt analysis. The main disadvantage is that any nonspecific products Labelling approaches formed in the PCR, including primer dimers are also detected. CYBR green

ssDNA -- unbound dye dsDNA -- bound dye >100 minimal fluorescence fold increase fluorescence

TaqM an -- Hydrolysis Probe

A TaqMan probe contains a fluorophore in close proximity to a quencher, such that the presence of the quencher blocks fluorescence. When bound to the target sequence, the probe is cleaved by the hn hn polymerase. The necessity of a probe to bind internal to the amplicon results in increased specificity. These assays can be multiplexed, but have the disadvantages of difficult design and TAQ-man probes fluor quencher cost of the dual labeled probe and the inability to perform a melt analysis.

Extension continues

Hybridization probes

Two labeled probes must bind within the amplicon to generate a fluorescence signal as energy is FRET transferred from the donor fluorophore to the acceptor fluorophore, a process known as hn hn fluorescence resonance energy transfer (FRET). These assays can be highly specific since two primers and two probes must bind. In addition, detection via hybridization probes is not dependent on a hydrolysis reaction, and thus, a melt analysis can be conducted. These assays can also be FRET probes multiplexed.

donor acceptor Monitor acceptor fluorescence

Figure 2. Figure X. Schematic of SYBR Green I, TaqMan, and hybridization probe fluorogenic detection approaches for real-time PCR.