Understanding Real-Time PCR and DNA Measurement Techniques
Explore the principles of real-time PCR, Ct values, sensitivity, and measuring DNA quantities using fluorescent reagents like SYBR Green I. Learn about copy number vs. Ct standard curves and the methods for quantifying DNA in a PCR reaction. Discover the significance of cycle numbers and how they relate to DNA amplification.
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Presentation Transcript
WHAT IF YOU STARTED WITH EIGHT TIMES LESS DNA TEMPLATE THAN I DID? YOU D ONLY HAVE 125,000 COPIES RIGHT NOW AT CYCLE 25 IMAGINING REAL-TIME PCR AND YOU D REACH 1,000,000 COPIES EXACTLY THREE CYCLES LATER THAN I WOULD! 5000000 4500000 4000000 3500000 MEASURING QUANTITIES 3000000 2500000 2000000 1500000 1000000 500000 0 0 5 10 15 20 25 30 35 40
WE DESCRIBE THE POSITION OF THE LINES WITH A VALUE THAT REPRESENTS THE CYCLE NUMBER WHERE THE TRACE CROSSES AN ARBITRARY THRESHOLD. IMAGINING REAL-TIME PCR THIS IS CALLED THE CT VALUE . CT VALUES ARE DIRECTLY RELATED TO THE STARTING QUANTITY OF DNA, BY WAY OF THE FORMULA: QUANTITY = 2^CT Ct Values: 5000000 4500000 4000000 3500000 MEASURING QUANTITIES 25 23 3000000 28 2500000 2000000 1500000 1000000 500000 0 0 5 10 15 20 25 30 35 40
LETS RECAP IMAGINING REAL-TIME PCR 1 unit Ct=25 4 units Ct=23 1/8 unit Ct=28 5000000 4500000 4000000 3500000 MEASURING QUANTITIES 3000000 2500000 2000000 1500000 1000000 500000 0 0 5 10 15 20 25 30 35 40
HOW SENSITIVE IS REAL-TIME PCR? IMAGINING REAL-TIME PCR Copy Number vs. Ct - Standard Curve 40 35 30 y = -3.3192x + 39.772 R2 = 0.9967 25 Ct 20 15 10 5 0 0 1 2 3 4 5 6 7 8 9 10 11 Log of copy number (10n) MEASURING ABSOLUTE QUANTITIES TYPICALLY ABOUT 100 COPIES IS AROUND THE MINIMUM AMOUNT. ONE HUNDRED COPIES OF A 200-BP GENE IS EQUIVALENT TO JUST TWENTY ATTOGRAMS (2 X 10-17 G) OF DNA!
PART 3: HOW DO WE ACTUALLY MEASURE DNA?
HOW DO WE MEASURE DNA IN A PCR REACTION? WE USE REAGENTS THAT FLUORESCE IN THE PRESENCE OF AMPLIFIED DNA! THE REAGENTS BIND TO DOUBLE- STRANDED DNA AND EMIT LIGHT WHEN ILLUMINATED WITH A SPECIFIC WAVELENGTH. SYBR GREEN I DYE IS THE MOST COMMONLY REAGENT.
SYBR GREEN I BINDS TO DOUBLE- STRANDED (DS) DNA 5 3 5 3 WHEN EXCITATION LIGHT IS APPLIED, BOUND SYBR GREEN I FLUORESCES INTENSE GREEN LIGHT. Extension Taq 5 ID ID 3 ID ID ID 5 3 Taq Apply Excitation Wavelength WHEREAS, SYBR GREEN THAT IS NOT BOUND TO DNA DOES NOT FLUORESCE Taq ID 5 3 ID ID ID ID FLUORESCENCE IS PROPORTIONAL TO THE AMOUNT OF DSDNA IN THE SAMPLE ID ID ID ID Taq 3 Repeat
REAL-TIME PCR INSTRUMENTS CONSIST OF THREE MAIN COMPONENTS: WHAT TYPE OF INSTRUMENTS ARE USED WITH REAL- TIME PCR? 1. THERMAL CYCLER (PCR MACHINE) 2. OPTICAL MODULE (TO DETECT FLUORESCENCE IN THE TUBES DURING THE RUN) 3. COMPUTER (TO TRANSLATE THE FLUORESCENCE DATA INTO MEANINGFUL RESULTS)
