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PCR-Presentation (Compatibility Mode)

Polymerase Chain Reaction (PCR) is an in vitro technique for amplifying specific DNA sequences, revolutionized by the discovery of thermostable Taq Polymerase in 1985. The PCR process involves a cycle of denaturation, primer annealing, and DNA synthesis, utilizing components such as DNA templates, primers, and enzymes. PCR has numerous applications, including diagnostics, genome mapping, and forensic analysis, and offers advantages like speed, sensitivity, and reproducibility.

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18 views24 pages

PCR-Presentation (Compatibility Mode)

Polymerase Chain Reaction (PCR) is an in vitro technique for amplifying specific DNA sequences, revolutionized by the discovery of thermostable Taq Polymerase in 1985. The PCR process involves a cycle of denaturation, primer annealing, and DNA synthesis, utilizing components such as DNA templates, primers, and enzymes. PCR has numerous applications, including diagnostics, genome mapping, and forensic analysis, and offers advantages like speed, sensitivity, and reproducibility.

Uploaded by

missshahnaz10
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Polymerase Chain Reaction

Introduction
• PCR, polymerase chain reaction, is an in-
vitro technique for amplification of a region
of DNA whose sequence is known or which
lies between two regions of known
sequence
• Before PCR, DNA of interest could only be
amplified by over-expression in cells and
this with limited yield
• 1966, Thomas Brock discovers Thermus
Aquaticus, a thermostable bacteria in the
hot springs of Yellowstone National Park
• 1983, Kary Mullis postulated the concept of
PCR ( Nobel Prize in 1993)
• 1985, Saiki publishes the first application of
PCR ( beta-Globin)
• 1985, Cetus Corp. Scientists isolate
Thermostable Taq Polymerase (from
T.Aquaticus), which revolutionized PCR
Reaction Components
• DNA template
• Primers
• Enzyme
• dNTPs
• Mg2+
• buffers
1- DNA template
• DNA containing
region to be
sequenced
• Size of target DNA
to be amplified : up
to 3 Kb
2- Primers
• 2 sets of primers
• Generally 20-30
nucleotides long
• Synthetically
produced
• complimentary to the
3’ ends of target DNA
• not complimentary to
each other
Primers (ctnd)
• Not containing inverted repeat sequences to
avoid formation of internal structures
• 40-60% GC content preferred for better
annealing
• Tm of primers can be calculated to
determine annealing T0
• Tm= .41(%G+C) + 16.6log(J+) + 81.5
where J+ is the concentration of monovalent
ions
3-Enzyme
• Usually Taq Polymerase or anyone of the
natural or Recombinant thermostable
polymerases
• Stable at T0 up to 950 C
• High processivity
• Taq Pol has 5’-3’ exo only, no proofreading
The PCR Cycle
• Comprised of 3 steps:
- Denaturation of DNA at 950C
- Primer hybridization ( annealing) at 40-
500C
- DNA synthesis ( Primer extension) at
720C
Standard thermocycle
RT-PCR
• Reverse Transcriptase PCR
• Uses RNA as the initial template
• RNA-directed DNA polymerase (rTh)
• Yields ds cDNA
Detection of amplification
products
• Gel electrophoresis
• Sequencing of amplified fragment
• Southern blot
• etc...
Applications
• Genome mapping and gene function
determination
• Biodiversity studies ( e.g. evolution studies)
• Diagnostics ( prenatal testing of genetic
diseases, early detection of cancer, viral
infections...)
• Detection of drug resistance genes
• Forensic (DNA fingerprinting)
Advantages
• Automated, fast, reliable (reproducible)
results
• Contained :(less chances of contamination)
• High output
• Sensitive
• Broad uses
• Defined, easy to follow protocols
References
• Fundamentals of Biochem ( Voet, Voet,
Pratt)
• Molecular Cell Biology ( Lodish, Darnell..)
Next Steps
• Summarize any actions required of your
audience
• Summarize any follow up action items
required of you

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