PCR With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies of the original DNA molecule.

PCR Components DNA nucleotides, the building blocks for the new DNA Template DNA, the DNA sequence that you want to amplify Primers, single-stranded DNAs between 20 and 30 nucleotides long that are complementary to a short region on either side of the template DNA Thermostable DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA. Buffer to maintain optimal pH for the enzyme to function

The cycling reaction There are three major steps in a PCR cycle, which are repeated for 20 to 40 cycles. This is done on an automated Thermo Cycler, which can heat and cool the reaction tubes in a very short time. Denaturation at around 94°C : During the denaturation, the double strand melts open to single stranded DNA. Annealing (between 50-60°C) : Extension at around 72°C : The polymerase adds dNTP's complementary to the template.

PCR Denature (heat to 95oC) Lower temperature to 56oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs

PCR is performed in a tube and when the reaction is complete the products of the reaction (the amplified DNA fragments) are analyzed and visualized by gel electrophoresis

Every cycle results in a doubling of the number of strands DNA present After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers The result is a dramatic amplification of a the DNA that exists between the primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 1012

PCR PCR tube THERMOCYCLER

Components of PCR Template DNA Flanking Primers Thermo-stable polymerase-Taq Polymerase dNTP (dATP, dTTP, dCTP, dGTP) PCR Buffer-Tris +Divalent cation(mg++) Add components to PCR tube Start run in Thermocyler

Typical Reaction 5 μl 10 X PCR buffer + mg 1 μl 200 μM dNTP 0.5 μl 50 μM Left Primer 0.5 μl 50 μM Right Primer 0.5 μl Taq Polymerase (5 Units/ μl) 10 μl DNA template 32.5 μl dH2O _____ ______________ 50 μl Total Vol

Master Mix preparation 1 sample --10 samples + extra tube _______ _____________________ 10 X PCR buffer + mg 5 μl 55 μl 200 μM dNTP 1 μl 11 μl 50 μM Left Primer 0.5 μl 5.5 μl 50 μM Right Primer 0.5 μl 5.5 μl Taq Polymerase (5 Units/ μl) 0.5 μl 5.5 μl dH2O 32.5 μl 358μl _______ _________ Total volume 40 μl 440 μl Distribute into 10 tubes + Add 10 μl sample to each tube

Visualization of PCR product

Qualitative +/- assay

Real Time PCR Real-time PCR, also called quantitative PCR or qPCR, can provide a simple method for determining the amount of nucleic acid target present in a sample . It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA .

Real Time PCR evolution

Real time PCR Uses various fluorescent dyes which react with the amplified product and can be measured by an instrument. Light hits the tube/vessel containing the PCR (once per cycle) Fluorescent dye(s) emit light corresponding to their spectral characteristics The emitted light is focused onto a detector The computer-software interface interprets the detector

Detection chemistries two basic chemistry classes 1-Probe based: TaqMan probes (fluorogenic 5' nuclease assay) TaqMan MGB probes Molecular beacons Adjacent probes/ (hybridization probes) Scorpions (labeled primer amplification) 2-Generic dye based: SYBR Green 1 dye Ethidium bromide Etc…

Real-Time PCR Real-Time PCR is identical to a simple PCR except that the progress of the reaction is monitored by a camera or detector in “real-time”. There are a number of techniques that are used to allow the progress of a PCR to be monitored. Each technique uses some kind of fluorescent marker which binds to the DNA. Hence as the number of gene copies increases during the reaction so the fluorescence increases. There is no need to run the PCR product out on a gel after the reaction.

Methods of monitoring DNA amplification in “real-time”: Fluorescent dyes Intercalating fluorescent dyes (e.g. SYBR green). These dyes fluoresce only when bound to double-stranded DNA. So as the number of copies of DNA increases during the reaction the fluorescence increases. The major disadvantage of using a dye such as this is the lack of specificity. This dye will report the amplification of any DNA not just your gene of interest.

Fluorescent probes An oligonucleotide probe containing a reporter fluorescent dye on the 5´ end and a quencher dye on the 3´ end. While the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye. If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5´ nuclease activity of Taq DNA polymerase as this primer is extended. This cleavage of the probe separates the reporter dye from the quencher dye, increasing the reporter dye signal. .

Real-Time PCR: 5‘- Nuclease Technology

Real-Time PCR: 5‘- Nuclease Technology Additional reporter dye molecules are cleaved from their respective probes with each cycle resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced

Nucleic Acid testing in Diagnosis

Real-Time PCR Applications Viral Quantitation Quantitation of Gene Expression Array Verification. Drug therapy efficacy / drug monitoring. Monitoring minimal residual disease Genotyping/Allelic Discrimination Pathogen detection Genetically Modified Organism (GMO) Detection