Pcr And Qpcr Flashcards
PCR
It is a laboratory technique allows for the rapid amplification of a specific DNA segment or a gene which can then be studied in greater detail.
Requires the enzyme Taq Polymerase.
▪ It is a chain reaction: DNA duplex formed at the end of one reaction cycle acts as the template for the next reaction cycle.
Each reaction cycle consist of 3 steps:desaturation,annealing(primers adhere to dns strands)extension which involves taq polymerase to build complementary strand
Can run 25-30 cycles
Components 0f pcr
-DNA template/sequence to be amplified.
- Taq Polymerase enzyme: is a thermostable DNA polymerase I
-Originally isolated from the thermophilic bacteria Thermus
aquaticus.
- Cannot initiate DNA synthesis de novo.
- Catalyzes the primer-dependent incorporation of nucleotides.
-Can add a nucleotide only onto a preexisting 3’-OH group.
- 5’→3’ synthesis of DNA strand
- dNTPs (deoxynucleotide triphosphate): adenine (dATP), cytosine (dCTP), guanine (dGTP), and thymine (dTTP).
▪ PCR primers are short, single-stranded segments of DNA that are complementary to the beginning and end of the target DNA sequence that will be amplified.
❖ Forward primers bind to the antisense strand (-) of the DNA that runs in 3′ to 5′ direction.
▪ Amplification of the antisense strand.
❖ Reverse primers bind to the sense strand (+) of the DNA that runs in the 5′ to 3′ direction.
▪ Amplification of the sense strand.
▪ Reaction buffer: PCR buffer provides a suitable chemical environment for activity of Taq polymerase.
➢ The buffer pH is usually between 8.0 and 9.5 required for the activity of Taq Polymerase.
➢Magnesium ions (Mg2+), which are essential cofactor for the polymerase activity and also helps in primer annealing.
What are thermocycylers
▪ Thermal cyclers or PCR machine.
▪ Regulate temperatures in a cyclical program
Visualisation of PCR-amplified DNA
▪ Agarose gel electrophoresis.
▪ Visualise different size of DNA regions that are amplified.
▪ Limitations:
• Presence of a “primer dimer” band • Excessive smearing, and
• Presence of nonspecific products.
Applications of pcr
1) Molecular Cloning and recombinant DNA technology
cloning gene of interest
2) Clinical diagnostics-
screening of genetic mutation in diseases like Cancer.
3) Forensic DNA Analysis/DNA fingerprinting/DNA profiling
Reverse transcription- quantitative Polymerase chain reaction (RT-qPCR)
▪ Technique to quantify mRNA level in cells for gene expression analysis.
▪ Two reactions:
1) Reverse transcription: synthesis of complementary DNA (cDNA) from mRNA by the enzyme
reverse transcriptase
2) quantitative PCR-The cDNA is then used as the template for the quantitative PCR.
▪ For example:
Change in expression of Gene ‘A’ in cancer cells as compared to non-cancerous cells.
Isolate mRNA from the two cell types Quantify by RT-qPCR.
Quantification would indicate if expression of Gene ‘A’ is affected in cancerous condition.
Reverse transcription-PCR
▪ Synthesis of DNA from an RNA template.
▪ Driven by RNA-dependent DNA polymerases, also known as reverse transcriptase.
▪ Primers: oligo-dT primers/Random hexamers/gene-specific primer.
▪ Oligo(dT) primers consist of a stretch of 12–18 deoxythymidines that anneal to poly(A) tails of eukaryotic mRNAs.
▪ Random hexamers
Reverse transcription pcr sequence
-primer annealing takes 5 min in 25 degrees
-dna polymerisation takes 30 min in 42 degrees
-enzyme inactivation in 85 degrees for 5min
Principle of qPCR
▪ Quantitative PCR: quantify the amount of target DNA present in the sample.
▪ Fluorescent dyes or probes: bind to target DNA during amplification.
▪ Fluorescence Signal emitted is proportional to the amount of target DNA present in the sample and can be quantified.
▪ qPCR can also be used to monitor the progress of amplification in real time by measuring the increase in fluorescence over time.
Fluorescence-based quantification in qPCR
1) Dye based method:
▪ Fluorescent DNA binding dye, binds double–stranded DNA.
▪ Increase in the number of DNA copies with each qPCR is monitored by the increase in
fluorescence throughout the cycle.
Example: SYBR green dye- intercalates nonspecifically into the double stranded DNA and emits fluorescence signal.
2) Probe based method also named as Taqman assay:
▪ Probes: synthetic oligonucleotides (designed for a specific DNA sequence) containing a reporter group that can be monitored using fluorescence detector.
▪ In qPCR, oligonucleotides modified with a 5’ fluorophore and a 3’ quencher are added to the reaction.
▪ Fluorescence of the fluorophore is suppressed by the quencher.
▪ During annealing, the probe hybridizes in a sequence-specific manner to the template DNA.
▪ During the extension reaction, the 5’→ 3’ exonuclease activity of Taq DNA polymerase degrades the hybridized probe, releasing quencher suppression and allowing fluorescence signal to be emitted.
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Taqman assay
2) Probe based method also named as Taqman assay:
▪ Probes: synthetic oligonucleotides (designed for a specific DNA sequence) containing a reporter group that can be monitored using fluorescence detector.
▪ In qPCR, oligonucleotides modified with a 5’ fluorophore and a 3’ quencher are added to the reaction.
▪ Fluorescence of the fluorophore is suppressed by the quencher.
▪ During annealing, the probe hybridizes in a sequence-specific manner to the template DNA.
▪ During the extension reaction, the 5’→ 3’ exonuclease activity of Taq DNA polymerase degrades the hybridized probe, releasing quencher suppression and allowing fluorescence signal to be emitted.
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▪ TaqMan Assays were named after the 1980s Pac-Man video game
▪ Exonuclease activity of Taq polymerase had similarities to the Pac-Man game.
▪ When Taq polymerase reaches a probe, it chews it to bits as it is creating a new amplicon.
Difference between SYBR green dye and tag man probe
Dye:
-dyebased
-less specific
-cost effective
-uses a flourosecnt dye that binds to double-stranded dna
-easier to use as it doesn’t require a probe design and synthesis
-can generate false positive signals
Probe
-probe based
More specific
-expensive
-uses a fluorgenic probe that’s specific to the target gene
-it can monitor the quantity of multiple qpcr products in a single reaction
-however it requires different probes for different sequences
Method of quantification
▪ qPCR/Real-time PCR machine combines the functions of a thermal cycler and a fluorimeter.
▪ Amplification (detected as Fluorescence signal) of a specific gene is recorded after each PCR cycle ▪ Real-time PCR data are often represented in a graph called an amplification plot.
Ct value
▪ The threshold cycle (Ct), is the specific cycle number at which the fluorescent signal for a sample exceeds the background fluorescence level.
▪ The point at which the fluorescence in a sample becomes measurable is called the Quantification Cycle (Cq) or crossing point.
• CT – threshold cycle
• Cp – crossing point
• TOP – take-off point
• Cq – quantification cycle
▪ CT values are inverse to the amount of target nucleic acid in the sample.
▪ Lower CT values (typically below 29 cycles) indicate high amounts of the target sequence.
▪ Higher CT values (above 38 cycles) mean lower amounts of your target nucleic acid sequence
Quantification and gene expression analysis
ΔΔCT or delta delta CT method for relative gene expression analysis.
▪ Relative gene expression: Analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample).
▪ Determines fold changes in expression between two samples.
▪ Variability: Quantity and quality of RNA extracted from multiple samples vary,
Sample degradation, extraction efficiency, and contamination.
▪ Proper normalization is essential for obtaining accurate gene expression studies
