Understanding Real-Time PCR: A Visual Journey from Basics to Application

Explore the fundamental principles of PCR and delve into the intricacies of real-time PCR through a visual narrative, tracing the DNA amplification process and its applications step by step in a PCR reaction tube up to cycle number 25. Witness the dynamic changes in DNA amounts through cycles, illustrating the essence of real-time PCR.

• PCR
• Real-time PCR
• DNA amplification
• Polymerase chain reaction
• Molecular biology

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Uploaded on Jul 22, 2024 | 1 Views

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1. HOW DOES REAL-TIME PCR WORK? TO BEST UNDERSTAND WHAT REAL- TIME PCR IS, LET S REVIEW HOW REGULAR PCR WORKS...

2. THE POLYMERASE CHAIN REACTION HOW DOES PCR WORK?? 5 3 3 5 d.NTPs Primers Thermal Stable DNA Polymerase Add to Reaction Tube Denaturation Annealing

3. 5 3 5 3 Extension Taq 5 3 5 3 Taq Extension Continued Taq 5 3 Taq 3 Repeat

4. 5 3 3 Cycle 2 3 5 3 3 5 3 4 Copies 3 3 5 3 3 5 3 3 3 5 3 Cycle 3 3 5 3 8 Copies 3 5 3 3 5 3 3 5 3 3 3

5. HOW DOES REAL-TIME PCR WORK? SO THAT S HOW TRADITIONAL PCR IS USUALLY PRESENTED. IN ORDER TO UNDERSTAND REAL-TIME PCR, LET S USE A THOUGHT EXPERIMENT , AND SAVE ALL OF THE CALCULATIONS AND FORMULAS UNTIL LATER MOST IMPORTANTLY, WE LL START BY IMAGINING THE PCR ITSELF, AND ONLY THEN WILL WE DRAW GRAPHS TO ILLUSTRATE WHAT S GOING ON.

6. TO UNDERSTAND REAL-TIME PCR, LETS IMAGINE OURSELVES IN A PCR REACTION TUBE AT CYCLE NUMBER 25 IMAGINING REAL-TIME PCR

7. IMAGINING REAL-TIME PCR WHAT S IN OUR TUBE, AT CYCLE NUMBER 25? A SOUP OF NUCLEOTIDES, PRIMERS, TEMPLATE, AMPLICONS, ENZYME, ETC. LET S ASSUME 1,000,000 COPIES OF THE AMPLICON RIGHT NOW.

8. IMAGINING REAL-TIME PCR WHAT WAS IT LIKE LAST CYCLE, 24? ALMOST EXACTLY THE SAME, EXCEPT THERE WERE ONLY 500,000 COPIES OF THE AMPLICON. HOW DID WE GET HERE? AND THE CYCLE BEFORE THAT, 23? ALMOST THE SAME, BUT ONLY 250,000 COPIES OF THE AMPLICON. AND WHAT ABOUT CYCLE 22? 125,000 COPIES OF THE AMPLICON.

9. IF WE WERE TO GRAPH THE AMOUNT OF DNA IN OUR TUBE, FROM THE START UNTIL RIGHT NOW, AT CYCLE 25, THE GRAPH WOULD LOOK LIKE THIS: IMAGINING REAL-TIME PCR 2000000 1800000 1600000 HOW DID WE GET HERE? 1400000 1200000 1000000 800000 600000 400000 200000 0 0 5 10 15 20 25 30 35 40

10. ? SO, RIGHT NOW WE RE AT CYCLE 25 IN A SOUP WITH 1,000,000 COPIES OF THE TARGET. IMAGINING REAL-TIME PCR WHAT S IT GOING TO BE LIKE AFTER THE NEXT CYCLE, IN CYCLE 26? 2000000 1800000 HOW DID WE GET HERE? 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 0 5 10 15 20 25 30 35 40

11. IMAGINING REAL-TIME PCR WHAT S IT GOING TO BE LIKE AFTER THE NEXT CYCLE, IN CYCLE 26? PROBABLY THERE WILL BE 2,000,000 AMPLICONS. AND CYCLE 27? MAYBE 4,000,000 AMPLICONS. AND AT CYCLE 200? IN THEORY, THERE WOULD BE 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000, 000,000,000,000,000 AMPLICONS OR 10^35 TONNES OF DNA TO PUT THIS IN PERSPECTIVE, THAT WOULD BE EQUIVALENT TO THE WEIGHT OF TEN BILLION PLANETS THE SIZE OF EARTH!!!! SO WHERE ARE WE GOING?

12. IMAGINING REAL-TIME PCR A CLUMP OF DNA THE SIZE OF TEN BILLION PLANETS WON T QUITE FIT IN OUR PCR TUBE ANYMORE. REALISTICALLY, AT THE CHAIN REACTION PROGRESSES, IT GETS EXPONENTIALLY HARDER TO FIND PRIMERS, AND NUCLEOTIDES. AND THE POLYMERASE IS WEARING OUT. SO WHERE ARE WE GOING? SO EXPONENTIAL GROWTH DOES NOT GO ON FOREVER!

13. IMAGINING REAL-TIME PCR IF WE PLOT THE AMOUNT OF DNA IN OUR TUBE GOING FORWARD FROM CYCLE 25, WE SEE THAT IT ACTUALLY LOOKS LIKE THIS: 5000000 4500000 SO WHERE ARE WE GOING? 4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 0 5 10 15 20 25 30 35 40

14. HOW CAN ALL THIS BE USED TO MEASURE DNA QUANTITIES?? IMAGINING REAL-TIME PCR 5000000 4500000 4000000 3500000 3000000 2500000 2000000 MEASURING QUANTITIES 1500000 1000000 500000 0 0 5 10 15 20 25 30 35 40

15. LETS IMAGINE THAT YOU START WITH FOUR TIMES AS MUCH DNA AS I DO PICTURE OUR TWO TUBES AT CYCLE 25 AND WORK BACKWARDS A FEW CYCLES. IMAGINING REAL-TIME PCR Cycle 25 Cycle Me You MEASURING QUANTITIES 23 250,000 1,000,000 24 500,000 2,000,000 25 1,000,000 4,000,000

16. SO, IF YOU STARTED WITH FOUR TIMES AS MUCH DNA TEMPLATE AS I DID IMAGINING REAL-TIME PCR THEN YOU D REACH 1,000,000 COPIES EXACTLY TWO CYCLES EARLIER THAN I WOULD! 5000000 4500000 4000000 3500000 3000000 MEASURING QUANTITIES 2500000 2000000 1500000 1000000 500000 0 0 5 10 15 20 25 30 35 40

17. WHAT IF YOU STARTED WITH EIGHT TIMES LESS DNA TEMPLATE THAN I DID? IMAGINING REAL-TIME PCR Cycle 25 Cycle Me You 25 1,000,000 125,000 MEASURING QUANTITIES 26 2,000,000 250,000 27 4,000,000 500,000 28 8,000,000 1,000,000

18. 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