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PCR Presentation

Polymerase chain reaction (PCR) is an in vitro technique for amplifying a specific region of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase and primers to selectively amplify the target region. The document outlines the key components of PCR including DNA template, primers, Taq polymerase enzyme, buffers, and cycling conditions. It also discusses applications such as genome mapping, diagnostics, and forensics. PCR is a sensitive and reliable method for amplifying DNA that has revolutionized research and clinical applications.

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258 views24 pages

PCR Presentation

Polymerase chain reaction (PCR) is an in vitro technique for amplifying a specific region of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase and primers to selectively amplify the target region. The document outlines the key components of PCR including DNA template, primers, Taq polymerase enzyme, buffers, and cycling conditions. It also discusses applications such as genome mapping, diagnostics, and forensics. PCR is a sensitive and reliable method for amplifying DNA that has revolutionized research and clinical applications.

Uploaded by

Usman Haider
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Polymerase Chain Reaction

Catherine Bangeranye
Biochem Seminar

Introduction
PCR, polymerase chain reaction, is an invitro 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 40500C
- DNA synthesis ( Primer extension) at 72 0C

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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