Revers transcriptase PCR princple

1. Reverse Transcriptase Polymerase
Chain Reaction
(RT-PCR)
Presented by:
Amany ELshamy

2. Outlines
• Introduction
• What is the Reverse Transcriptase PCR?
• Principle
• Reverse Transcriptase PCR Vs. traditional PCR
• Material
• Method
• Equipment
• Application
• Questions
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3. Introduction
• Reverse Transcription Polymerase Chain
Reaction (RT-PCR) is one of the polymerase
chain reaction (PCR) types.
• It is mainly used in molecular biology to
detect RNA expression.
3

4. What is the Reverse Transcriptase
PCR?
• RT-PCR is used to qualitatively detect gene
expression through creation of
complementary DNA (cDNA) transcripts from
RNA.
4

5. Reverse transcriptase PCR
Quantitative Reverse
Transcription
Polymerase chain
reaction is used when
the specific target RNA
amplified and detected
simultaneously.
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6. Reverse transcriptase PCR principle
• The RNA is first convert into complementary
DNA (cDNA) through the use of reverse
transcriptase before amplification.
• It is widely used for the evaluation of the gene
expression and viral load.
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7. Reverse transcriptase PCR principle
• Use mRNA as a template to produce cDNA;
“Reverse transcriptase enzyme”
• cDNA is then amplified to screen possible
mutations directly.
Reverse
transcriptas
e enzyme
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8. 8

9. Reverse Transcriptase PCR Vs.
traditional PCR
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10. Reverse Transcriptase PCR Method
• Reverse transcriptase is added to solution
containing:
RNA (sample)
dNTPs
Primers
Buffers
DNA polymerase
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11. Reverse Transcriptase PCR Method
• RNA is converted to cDNA.
• Then, the DNA sequence of interest is amplified
in three steps: (1) denaturation, (2) annealing, (3)
elongation.
• Assess the amount of RNA in the original sample
with gel electrophoresis which is directly
proportional to the concentration of RNA in the
original sample.
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12. Reverse Transcriptase PCR types
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13. Reverse Transcriptase PCR types
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14. Equipment
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15. Application
• Studying the genomes of viruses composed of
RNA ex; HIV, Influenza, COVID-19…etc.
• Measure a viral load and detect the presence
of infection.
• Genetic Disease Diagnosis.
• Cancer Detection.
• Research methods
15

16. Video
• https://www.youtube.com/watch?v=JGoGhp6
vcSE
• https://www.youtube.com/watch?v=iiXisgizkx
s
• https://www.youtube.com/watch?v=qmACKSv
FZpM
• https://www.youtube.com/watch?v=ThG_02
miq-4
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17. Qs
1. Reverse transcriptase produces DNA from RNA.
a) True
b) False
2. How many primers are used in the process of
reverse transcriptase amplification?
a) 1
b) 2
c) 3
d) 4
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18. Questions
3. From which organism is the enzyme reverse
transcriptase isolated?
a) Bacteria
b) Fungi
c) Virus
d) Prions
Explanation: viruses are retrovirus, human
papiloma virus, etc.
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19. Questions
4. Reverse transcription PCR uses _________.
(a) RNA as a template to form DNA
(b) mRNA as a template to form cDNA
(c) DNA as a template to form ssDNA
(d) All of the above
Sol:(b) mRNA as a template to form cDNA.
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