Rea eal-time time PCR CR th theory eory and d appl pplications ications By By Dal alia ia M. M. Sab abri, , PhD hD La Lab b Ma Mana nager ger , B , BRC, C, SCU CU Biotechnology Research Center, Suez Canal University, 11-11-2015
* PCR History * What is PCR? * Endpoint PCR Vs Real-time PCR * Real-time PCR * QPCR Detection Chemistry * Real-time PCR Instruments * Methods of Quantification * New technologies
PCR History 1985 PCR first published Late1980 ’s PCR used for quantification, but required electrophoresis to detect product at end-point of PCR Use of 5 ’ -> 3 ’ exonuclease activity of Taq polymerase to detect specific PCR 1991 activity (“TaqMan” approach). 1992 Discovery that EtBr (dsDNA probe) can be added to the PCR mix and that the fluorescence of EtBr will increase at each cycle of PCR due to product formation. No need to cool to room temperature or to open tubes to measure the increased EtBr fluorescence. First real-time experiments – in closed PCR tubes on UV- trans-illuminator. Higuchi and others at Roche and Chiron . 1993 First real-time PCR detection experiments to show utility for DNA quantitation. Improved EtBr detection (illumination, CCD camera detection). Higuchi and others at Roche . 1996 TaqMan detection methods used, instead of EtBr, for real-time detection of PCR. Improved specificity. Heid and others at ABI and Genentec. ABI introduces first real- time qPCR (“Sequence Detection System”) instrument 1996-7 (the ABI 7700). Since then Many more instrument manufacturers (Roche, BioRad, Stratagene, Corbett, Cepheid, MJ) and many more detection chemistries.
What is PCR? * The P olymerase C hain R eaction ( PCR ) is a revolutionary technology developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand, that results in generating thousands to millions of copies of a particular DNA sequence from a single copy or a few copies of a piece of DNA . *The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzmatic replication of the DNA.
The three-step PCR process 1. Denaturation step (Separating the Target DNA) DNA template is heated to more than 90 ºC , which separates the double- stranded DNA into two separate single strands.
The three-step PCR process 2. Annealing step (Binding Primers to the DNA Sequence) The reaction is cooled between 40 and 60 ºC . PCR oligonucleotides bind (anneal) to sequences on either side of the target DNA region, marking off the sequence for step three
The three-step PCR process 3. Extension step (Making a Copy) The temperature is increased to 72 ºC , nucleotides in the solution are added to the annealed primers by the DNA polymerase to create a new strand of DNA complementary to each of the single template strands. After completing the extension, two identical copies of the original DNA have been made.
PCR amplification curve
• Exponential - Exact doubling of product is accumulating at every cycle. - The reaction is very specific and precise. - All of the reagents are fresh and available, the kinetics of the reaction push the reaction to favor doubling of amplicon. • Linear (high variability) - As the reaction progresses, some of the reagents are being consumed in amplification process. - The reactions start to slow down and the PCR product is no longer being doubled at each cycle. • Plateau (End-point) -The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.
Endpoint PCR Vs Real-time PCR
Endpoint PCR Vs Real-time PCR Endpoint PCR Real-time PCR Measures the amount of accumulated PCR product at the Measures PCR amplification as it end of the PCR cycles occurs Reaction endpoint data collection Ct
Endpoint PCR Vs Real-time PCR Endpoint PCR Real-time PCR Semi-quantitative Quantitative Data collection during the Though comparing the intensity of exponential (log) phase makes the the amplified band on a gel to quantity of the PCR product is standards of a known directly proportional to the amount concentration. of template nucleic acid.
Endpoint PCR Vs Real-time PCR Endpoint PCR Real-time PCR Semi-quantitative Quantitative Data collection during the Though comparing the intensity of exponential (log) phase makes the the amplified band on a gel to quantity of the PCR product is standards of a known directly proportional to the amount concentration. of template nucleic acid.
Endpoint PCR Vs Real-time PCR Endpoint PCR Real-time PCR Applications Applications Amplification of DNA for: Gene expression quantification Sequencing Microarray verification Genotyping Quality control and assay validation Cloning Pathogen detection SNP genotyping Copy number variation MicroRNA Analysis Viral quantification
Endpoint PCR Vs Real-time PCR Endpoint PCR Real-time PCR Disadvantages: Advantages: - Low sensitivity. - Increased dynamic range of detection - Post-PCR processing. - No post PCR processing. - Low resolution and Poor Precision. - Detection is capable down to a 2-fold - Non-automated. change. - Collects data in the exponential growth -Size-based discrimination only & phase of PCR. results are not expressed as numbers. - An increase in reporter fluorescent signal is directly proportional to the number of -Ethidium bromide for staining is not very amplicons generated. quantitative. - The cleaved probe provides a record for amplified amplicons.
Real-time PCR
Real-time PCR *Real-time PCR combines PCR amplification and detection into a single step. *With Real-time PCR, fluorescent dyes are used to label PCR products during thermal cycling with the capacity to illuminate each sample with a beam of light of at least one specified wavelength . *Real-time PCR instruments measure the accumulation of fluorescent signal emitted by the excited fluorophore.
QPCR Detection Chemistry 1- Non-specific detection (DNA binding dyes): SYBR Green, BEBO, BOXTO, EvaGreen. 2- Specific detection (Probe based test): TaqMan, Molecular Beacons, Hybridization Probes (Scorpions, Amplifluor Primers, LUX Primers, FRET probe pairs).
SYBR Green I SYBR Green I dye binds to double- stranded DNA and emits fluorescence only when bound, DNA-dye-complex absorbs blue light (λ= 497 nm) and emits green light (λ= 520 nm). The stain can also binds to single-stranded DNA and RNA but with lower performance.
SYBR Green I SYBR Green I dye binds to double- stranded DNA and emits fluorescence only when bound, DNA-dye-complex absorbs blue light (λ= 497 nm) and emits green light (λ= 520 nm). The stain can also binds to single-stranded DNA and RNA but with lower performance. *Advantages: 1- Low cost assay. 2-Easy to design and set up. *Disadvantages: 1-Non specific system as it detects all ds DNA , in poorly designed PCR reaction primer-dimer product will be detected and quantified 2-Not adapted to multiplex .
TaqMan probe-based assay TaqMan probes consist of a fluorophore 5 ’ -end reporter attached to the of the oligonucleotide probe and a quencher at the 3 ’ -end. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source.
TaqMan probe-based assay TaqMan probes consist of a fluorophore 5 ’ -end reporter attached to the of the oligonucleotide probe and a quencher at the 1- 3 ’ -end. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source. 2- 3- 4-
TaqMan probe-based assay TaqMan probes consist of a fluorophore 5 ’ -end reporter attached to the of the oligonucleotide probe and a quencher at the 1- 3 ’ -end. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source. 2- Advantages: 1- very specific 2- Can be used in multiplex qPCR assays Disadvantages: 1- difficult to design 3- 2- More expensive than SYBR Green assay Applications: *Gene expression assays 4- *Determine the viral load in clinical specimens *Bacterial Identification assays *SNP genotyping *Verification of microarray results
Real-time PCR Instruments
Any Real-time PCR Instrument consists of two main components
Any Real-time PCR Instrument consists of two main components Thermal Cycler (PCR machine)
Any Real-time PCR Instrument consists of two main components Optical module ( to detect fluorescence in the tubes during the run) 1- Halogen lamp 2- Filter /detector : (5 dyes detectors) FAM/SYBR Green I, VIC/JOE, NED/TAMRA/Cy3, ROX/Texas Red, Cy5 3- CCD camera
Methods of Quantification Absolute Quantification Relative Quantification Quantification of unknown samples based This method analyze changes in gene on a known quantity. First you create expression in a given sample relative to a standard curve; then you compare another reference sample (such as an unknowns to the standard curve and untreated control sample). extrapolate a value.
Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (exceeds background level). Threshold is the point of detection. Rn Cycle-Threshold (Ct) Cycle cycle at which sample crosses threshold
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