Recombinant DNA Technology''

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Recombinant DNA Technology..

• BTEC3301

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Polymerase Chain Reaction (PCR)

• Introduction
• The Polymerase Chain Reaction (PCR) provides an
  extremely sensitive means of amplifying small
  quantities of DNA.
• Kary Mullins won the Nobel Prize in 1993 for
  development of this novel technique.

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Polymerase Chain Reaction (PCR)

• Introduction
• The technique was made possible by the discovery
  of Taq polymerase, the DNA polymerase that is
  used by the bacterium Thermus auquaticus ,
  discovered in hot springs.

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Polymerase Chain Reaction (PCR)

• Introduction
• The use of thermostable DNA polymerases was an
  important breakthrough since this enzyme does not
  denature at the temperatures( 950C) used to
  cause denaturation of the DNA.

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Polymerase Chain Reaction (PCR)

• Introduction
• The most well-known of these thermostable DNA
  polymerases is Taq.
• This enzyme has a molecular size of 94kD and an
  optimum reaction temperature of 75-80oC.

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Polymerase Chain Reaction (PCR)

• Introduction
• Amplification means making multiple identical
  copies (replicates) of a DNA sequence.
• PCR method of DNA amplification has proved very
  important in recombinant DNA technology and is
  used in a range of applications in medicine and
  forensic science .

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Polymerase Chain Reaction (PCR)

• What is PCR?
• It is used to amplify a specific DNA (target)
  sequence lying between known positions (flanks)
  on a double-stranded DNA molecule.
• The amplification process is mediated by
  oligonucleotide primers that, typically, are
  20-30 nucleotides long.

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Polymerase Chain Reaction (PCR)

• What is PCR?
• The primers are single-stranded DNA.
• Primers anneal to the flanking regions by
  complementary-base pairing (GC and AT) using
  hydrogen bonding.

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Polymerase Chain Reaction (PCR)

• The amplified product is known as an amplicon.
• Generally, PCR amplifies smallish DNA targets
  100-1000 base pairs (bp) long.

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Polymerase Chain Reaction (PCR)

• Material required in PCR process
• Thermal cycler (PCR machine, available in
  different specificity and models).

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Polymerase Chain Reaction (PCR)

• PCR mix for a reaction has the following
• sample DNA with a target sequence
• thermostable DNA polymerase (Taq is commonly
  used)
• two oligonucleotide primers which are
  complementary to the sequence flanking the target
  sequence.

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Polymerase Chain Reaction (PCR)

• deoxynucleotide triphosphates (dNTPs)
• reaction buffer containing magnesium ions and
  other components. HomeworkFunction of Magnesium
  for Quiz!!!

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Polymerase Chain Reaction (PCR)

• Steps in PCR reactions
• 1.    Heat denaturation
• A DNA molecule carrying a target sequence is
  denatured by heat at 90-95oC. The two strands
  separate due to breakage of the hydrogen bonds
  holding them together.

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Polymerase Chain Reaction (PCR)

• 2.   Primer annealing
• Primers are added to the dissociated target
  strands and incubated together first at a
  temperature that allows the primers to hybridize
  to the target strands (usually somewhere between
  40 and 55 oC).

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Polymerase Chain Reaction (PCR)

• 3. Primer extension
• DNA polymerase is then added and complementary
  strands are synthesized at a temperature of
  60-75oC. The polymerase causes synthesis of new
  material in the 5' to 3' direction away from each
  of the primers.

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Polymerase Chain Reaction (PCR)

• This allows a first round of synthesis to occur
  on each of the DNA templates.
• Following primer extension, the mixture is heated
  (again at 90-95oC) to denature the molecules and
  separate the strands and the cycle repeated

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Polymerase Chain Reaction (PCR)

• Each new strand then acts as a template for the
  next cycle of synthesis.
• Amplification (replication) proceeds at an
  exponential (logarithmic) rate (amount of DNA
  produced doubles at each cycle).

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Polymerase Chain Reaction (PCR)

• A thermal cycler (a machine that automatically
  changes the temperature at the correct time for
  each of the stages and can be programmed to carry
  out a set number of cycles) is used for a PCR
  reaction.

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Polymerase Chain Reaction (PCR)

• The amplified product can be detected using gel
  electrophoresis to view the band containing DNA
  fragments of a particular size containing the
  gene of interest in the original starter DNA
  sample.

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• Diagrammatic steps in PCR process (Read summary)
• Three major steps in PCR
• 1. Template denaturation
• 2. Primer annealing
• 3. Primer extension

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Home work for next class!!

• Summarize the steps in PCR process
• The oligonucleotides serve as primers for DNA
  polymerase and the denatured strands of the large
  DNA fragment serves as the template.
• This results in the synthesis of new DNA
  strands which are complementary to the parent
  template strands.
• After each cycle the newly synthesized DNA
  strands can serve as templates in the next cycle.

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Polymerase Chain Reaction (PCR)

• Diagrammatic steps in PCR process
• PCR amplification is achieved by using
  oligonucleotide primers.

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Polymerase Chain Reaction (PCR)

• Diagrammatic steps in PCR process

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Fig. 3.8 The Polymerase Chain Reaction
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Go to Animation courtesy of the following
websitesReferences

• http//faculty.plattsburgh.edu/donald.slish/PCRmov
  .html (Animation)
• http//www.accessexcellence.org/RC/AB/IE/PCR_Xerox
  ing_DNA.html
• http//www.people.virginia.edu/rjh9u/pcranim.html
  ( PCR Animation)
• http//www.escience.ws/b572/L3/L3.htm (PCR
  Animation)
• http//homepages.strath.ac.uk/dfs99109/BB211/Reco
  mbDNAtechlect4.html
• http//en.wikipedia.org/wiki/Polymerase_chain_reac
  tion
• http//www.escience.ws/b572/L3/L3.htm
• http//allserv.rug.ac.be/avierstr/principles/pcra
  ni.html