appl pplications ications By By Dal alia ia M. M. Sab abri, - - PowerPoint PPT Presentation
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,
By By Dal alia ia M.
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
1985 PCR first published Late1980’s PCR used for quantification, but required electrophoresis to detect product at end-point of PCR 1991 Use of 5’-> 3’ exonuclease activity of Taq polymerase to detect specific PCR 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
1996 TaqMan detection methods used, instead of EtBr, for real-time detection of PCR. Improved specificity. Heid and others at ABI and Genentec. 1996-7 ABI introduces first real-time qPCR (“Sequence Detection System”) instrument (the ABI 7700). Since then Many more instrument manufacturers (Roche, BioRad, Stratagene, Corbett, Cepheid, MJ) and many more detection chemistries.
* The Polymerase Chain Reaction (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
reaction for DNA melting and enzmatic replication
DNA template is heated to more than 90 ºC, which separates the double- stranded DNA into two separate single strands.
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 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.
the reaction push the reaction to favor doubling of amplicon.
consumed in amplification process.
longer being doubled at each cycle.
and if left long enough, the PCR products will begin to degrade.
Endpoint PCR Real-time PCR Measures the amount of accumulated PCR product at the end of the PCR cycles Measures PCR amplification as it
Reaction endpoint data collection
Ct
Endpoint PCR Real-time PCR Semi-quantitative Though comparing the intensity of the amplified band on a gel to standards of a known concentration. Quantitative Data collection during the exponential (log) phase makes the quantity of the PCR product is directly proportional to the amount
Endpoint PCR Real-time PCR Semi-quantitative Though comparing the intensity of the amplified band on a gel to standards of a known concentration. Quantitative Data collection during the exponential (log) phase makes the quantity of the PCR product is directly proportional to the amount
Endpoint PCR Real-time PCR Applications Amplification of DNA for: Sequencing Genotyping Cloning
Applications
Gene expression quantification Microarray verification Quality control and assay validation Pathogen detection SNP genotyping Copy number variation MicroRNA Analysis Viral quantification
Endpoint PCR Real-time PCR
Disadvantages:
results are not expressed as numbers.
quantitative. Advantages:
change.
phase of PCR.
directly proportional to the number of amplicons generated.
amplified amplicons.
*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.
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 dye binds to double-
stranded DNA and emits fluorescence
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 dye binds to double-
stranded DNA and emits fluorescence
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 probes consist of a fluorophore reporter attached to the 5’-end
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 probes consist of a fluorophore reporter attached to the 5’-end
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.
1- 2- 3- 4-
TaqMan probes consist of a fluorophore reporter attached to the 5’-end
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.
Advantages: 1- very specific 2- Can be used in multiplex qPCR assays Disadvantages: 1- difficult to design 2- More expensive than SYBR Green assay Applications:
*Gene expression assays *Determine the viral load in clinical specimens *Bacterial Identification assays *SNP genotyping *Verification of microarray results
1- 2- 3- 4-
Any Real-time PCR Instrument consists of two main components
Any Real-time PCR Instrument consists of two main components
Thermal Cycler (PCR machine)
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
Any Real-time PCR Instrument consists of two main components
Absolute Quantification Relative Quantification
Quantification of unknown samples based
a standard curve; then you compare unknowns to the standard curve and extrapolate a value. This method analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample).
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.
Cycle-Threshold (Ct) cycle at which sample crosses threshold
Rn
Cycle
Melting curve:
The melting curve analysis is based on the Tm, in which half of the DNA molecules are in the form of single strand and the other half in the form of a double helix. The Tm is dependent on DNA composition, since each amplified fragment has a specific size and base composition, they can be identified by melting curve analysis. FIGURE : Melting curve analysis for HBV genotyping. A (68 bp), D (119 bp) and F (97 bp)
F IN PATIENTS FROM A GENERAL HOSPITAL IN SOUTHERN BRAZIL. Arq. Gastroenterol., São Paulo , v. 50, n. 3, p. 219-225, Sept. 2013 .
Why we should collect results during exponential phase?
Plateau effect
Rn Exponential Phase
Digital PCR works by partitioning a sample into many individual real- time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.
QX100™ Droplet Digital Bio-Rad
(A) Samples partitioning into 20,000 nanoliter- sized droplets (B) PCR on a thermal cycler (C) The detector reads each droplet on turn at the rate 1000 droplet per sec