PCR (Polymerase Chain Reaction) Explained

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Polymerase Chain Reaction (PCR), is a genetic copying process used in biotechnology. This video covers what PCR is, what it is used for, the reagents of PCR, the steps of PCR, and how RT-qPCR is used in Covid testing.

PCR REAGENTS:
DNA Sample: Contains the sequence of interest to be copied
Taq Polymerase: Enzyme that will build the new DNA strands
Deoxynucleoside Triphosphatases: Building blocks for the new strands
Primers: Single stranded sequences that bracket the sequence of interest
Buffer Solution: Maintains optimal conditions for the reaction to occur
Magnesium Cofactors: Helps the primers to adhere at the correct site, and it helps Taq Polymerase to function optimally

PROCESS OF PCR:
PCR has 3 phases: Denaturation, Annealing and Extension. During the Denaturation Phase, the DNA strands are separated using heat, creating two new template strands. During the Annealing Phase, primers adhere to the start of the sequence of interest, marking where the Taq Polymerase should begin building. During the Extension Phase, the Taq Polymerase builds new complementary DNA strands, using the template strands as a guide. At the end of the cycle, the amount of DNA has doubled.

HOW IS PCR USED IN COVID TESTING?
PCR testing for the virus uses a modified version of PCR called RT-qPCR (reverse transcription quantitative PCR). This type of PCR uses a naturally occurring enzyme called Reverse Transcriptase to convert single stranded viral RNA into double stranded DNA suitable for PCR. Dye is added to the reaction that will fluoresce in the presence of viral DNA. As the cycles progress and the amount of viral DNA (if any) continues to double, the sample will glow brighter and brighter. A computer tracks the level of fluorescence and labels a sample as positive if the brightness exceeds a particular threshold. The two major types of fluorescent dye methods are SYBR Green, and Taqman Probe Assays.

0:00-0:16 Introduction
0:17-0:56 What is PCR?
0:57-1:35 Uses of PCR: Forensics, Agriculture & Medicine
1:35-1:46 Reagents of PCR: Overview
1:46-1:53 DNA Sample in PCR
1:53-2:16 Taq Polymerase in PCR
2:16-2:32 DNTPs in PCR
2:32-2:54 PCR Primers
2:55-3:16 PCR Buffer
3:16-3:29 PCR Magnesium Cofactors
3:30-3:53 PCR vs DNA Replication
3:53-4:16 Denaturation Phase of PCR
4:17-5:41 Annealing Phase of PCR
5:41-6:22 Extension Phase of PCR
6:23-6:50 Exponential Growth
6:50-7:16 RT-qPCR in Covid Testing
7:16-8:02 Reverse Transcription in RT-qPCR for Covid Testing
8:02-8:52 Quantitative PCR for Covid Testing
8:52-9:52 SYBR Green and TaqMan Probe Assays in Covid Testing
9:52-10:49 False Positives vs False Negatives

USEFUL SOURCES FOR FURTHER READING:
PCR:

DNA Replication:

Taq Polymerase:

PCR Covid Testing:

CITATION:
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