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Real Time PCR
The document provides an overview of real-time PCR (Polymerase Chain Reaction), detailing its significance, processes, and applications in biotechnology. It covers the chemistries used, such as DNA binding dyes and probes, and explains absolute and relative quantification methods along with practical precautions. Additionally, it highlights real-time PCR applications including viral quantitation, gene expression analysis, and pathogen detection.
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Real Time PCR
- 1. Real Time PCR AshikhSeethy Senior Resident and PhD Scholar Dept of Biochemistry AIIMS- New Delhi
- 2. Studying Gene Expression RNAextraction and quantification cDNA synthesis and confirmation of synthesis End Point PCR or Real Time PCR
- 3. Overview: ◉ What isReal Time PCR? ◉ Why Real Time PCR? ◉ Chemistries used in Real Time PCR ◉ The process of Real Time PCR ◉ Absolute and relative quantification ◉ Melting curve ◉ Precautions ◉ Applications
- 4. What is RealTime PCR? Why Real Time PCR?
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- 6. Problems with detectionin the plateau phase of PCR
- 7. Problems with end-pointdetection
- 8. Problems with end-pointdetection Poor precision Low sensitivity Low resolution Non - automated Size-based discrimination only Ethidium bromide for staining
- 9. Alternative? Real Time PCR RealTime PCR uses fluorescence detecting thermocyclers to amplify specific nucleic acid sequences and measure their concentrations simultaneously
- 10. Chemistries in RealTime PCR
- 11. Chemistries in RealTime PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers
- 12. Chemistries in RealTime PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers
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- 14. SYBR Green ILimitations: ◉ Detection of non-specific ds reaction products ◉ Increased background or false positives ◉ Inhibition of the PCR reaction Other dyes: ◉ SYTO 9 ◉ Evagreen
- 15. Chemistries in RealTime PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers
- 16. Taq Polymerase +PacManTaq Polymerase + PacMan
- 17. Chemistries in RealTime PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers
- 19. Chemistries in RealTime PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers
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- 21. The Process ofReal Time PCR
- 22. One Step andTwo Step Real Time PCR
- 23. Initial denaturation: 94oCfor 15 minutes - 1 cycle, followed by 40 cycles of: Denaturation at 94.0oC for 15 seconds Annealing at 55.8oC for 45 seconds Extension at 72.0oC for 45 seconds Sl No. Reagent Quantity/Reaction 1. cDNA 2 µL 2. Maxima SYBR Green/ROX qPCR Master Mix 6 µL 3. Forward primer – 25 pmol/µL 0.3 µL 4. Reverse primer – 25 pmol/µL 0.3 µL 5. Nuclease free water 12 µL
- 24. Controls in RealTime PCR: ◉ Non-Template Control/ Negative Control ◉ Minus RT control detects DNA contamination ◉ No amplification control ◉ Positive control- exogenous/ endogenous ◉ Internal/ Normalisation control ◉ Passive reference dye
- 25. Carrying Out RealTime PCR:
- 26. Absolute and RelativeQuantification
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- 28. Relative Quantification- ComparativeCT method ◉ Relative concentration of the gene of interest (GOI) in unknown samples is compared to a calibrator, or control sample ◉ Examples: 1. Drug treated cell lines and untreated cell lines 2. Patient samples and healthy controls ◉ Differences in Ct value between an unknown sample and control are expressed as fold-changes relative to the control sample (ΔCt)
- 29. Relative Quantification- ComparativeCT method ◉ To control the differences in RNA isolation and in the efficiency of the reverse transcription from sample to sample and experiment to experiment ↓ ◉ Normalization with reference gene, typically a gene whose expression is constant in both the control and experimental samples like 18S rRNA, GAPDH and ACTB. i.e., ΔΔCt = (CtTarget-CtACTB)Cases-(CtTarget-CtACTB)Controls ◉ Fold change = 2-ΔΔCt
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- 33. Precautions: Product length: ◉ 70–150 bp for probe-based chemistries ◉ 100– 300 bp for SYBR Green Primers incorporating exon-exon junctions RNA extraction and quantification cDNA synthesis and confirmation of synthesis Real Time PCR
- 34. Precautions: Avoid primer dimerizationand misfolding: ◉ GC clamps ◉ Inverted repeats Melting Curve Analysis Controls RNA extraction and quantification cDNA synthesis and confirmation of synthesis Real Time PCR
- 35. Precautions: SYBR Green Iis light sensitive Avoid marking on PCR tubes Prepare a cocktail Pipetting RNA extraction and quantification cDNA synthesis and confirmation of synthesis Real Time PCR
- 36. Applications of RealTime PCR
- 37. Applications Viral Quantitation Quantitationof Gene Expression Array Verification Pathogen detection Drug Therapy Efficacy Genotyping
- 38. Genotyping High resolution meltcurve analysis
- 39. Genotyping Minor Groove BindingTaqman Probes Locked Nucleic Acid Probes
- 41.
Editor's Notes
- #15 absorbs blue light (λmax = 497 nm) and emits green light (λmax = 520 nm)
- #17 Several different fluorophores (e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET) and quenchers (e.g. tetramethylrhodamine, acronym: TAMRA) are available
- #28 Concentration is measured by A260 and converted to the number of copies using the molecular weight of the DNA or RNA.