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Quantitative Polymerase Chain Reaction in Real‐Time (qPCR)
Natalia Ogorodnikova
• Introduction• PCR Principle • Application• qPCR • My example• Interactive element
A Couple Words about Genetics
Central Dogma of Genetics
Purpose of PCR is to have a huge ammount of target DNA
PCR Principle
/www.biologydiscussion.com
PCR Principle_Video
• https://www.youtube.com/watch?v=iQsu3Kz9NYo
Questionary
1. What happens during step1 at 94’C?2. What happens during step2 at 60’C?3. What happens during step3 at 72’C?4. How many cycles are required for PCR
amplification?
PCR HISTORY
Let’s prepare PCR
• DNA sample• Primers• DNA Polymerase Mg2+
• Deoxynucleotides (A, T, G and C)• Polymerase buffer
PCR Application
What applications does PCR have?
PCR Application
• Medical Application– Genetic testing, Mutation Analysis(Breast Cancer Test,
Hyperbilirubinemia, Prenatal Testing)
– Virus identification (HIV, tuberculosis)– Forensic medicine (genetic fingerprinting)– DNA paternity
PCR Application
• Research Application– In DNA Sequencing– In DNA cloning– Phylogenic analysis– Gene expression study
PCR Analitics
Or Real‐Time PCR
Fluorescent Probes for q PCR Analysis
• Non‐specific detection: Sybr Green dye, BEBO, BOXTO, EvaGreen
• Specific Detection: TaqMan Probe, Molecular Beacon, Scorpion primer….
Specific Detection
• Molecular Beacon
• Hybridization Probe
Data Analysis_ Amplification Curves
• Amplification Curve Analysis
Data Analysis_Analysis_Melting Curve Analysis
What was amplified?
• Melting Curve Analyses
Data Analysis_Standard Curve Analysis
Quantification!!!
My Example_miRNA122 Quantification
My Project: Synthesis of micro RNA inhibitors.I use qPCR for microRNA quantification in cells
Data Analysis
Data Analysis
Results
ΔΔCt=(Ct target gene- Ct reference gene)treated-(Ct target gene-Ct reference gene)control
DA8 5µM -2,46 **10µM -4,60 *20µM -5,35 ***
DA40 5µM -2,87 ***10µM -4,30 **20µM -27,87 ***
miR122 Change due to treatment 2^-∆∆Ct
Significance :p‐value‐*p<0,05; **p<0,01; ***p<0,005)
Puzzle
• Gene target chose, Primer Design and Primer Order
• DNA Extraction or RNA extraction (for RNA extraction ‐ cDNA synthesis)
• PCR preparation (DNA Sample, Primers, Deoxynucleotides (A, T, G and C), DNA Polymerase(Mg2+), Dye)
• PCR run• Data Analysis (delta Ct method)
Take‐Home Message
1. Gene Target and primer design. Choose thestrategy of detection in qPCR
2. Select controls and standards for reaction3. Sample preparation (DNA or RNA, sourse of
sample)4. Chose right DNA polymerase (long fragments,
precise amplification of short fragments, 5‘‐3‘ endonuclease activity)