Polymerase Chain Reaction

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Polymerase Chain Reaction

• Group 3
• Mitika Patel
• Sheena Jain
• Poonum Bharal
• Aditi Dhakar

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• It is hard to exaggerate the impact of the
  polymerase chain reaction. PCR, the quick, easy
  method for generating unlimited copies of any
  fragment of DNA, is one of those scientific
  developments that actually deserves timeworn
  superlatives like "revolutionary" and
  "breakthrough." 
• - Tabitha M. Powledge

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Purpose of PCR

• Amplify specific nucleic acids in vitro
  (Xeroxing DNA)
• PCR will allow a short stretch of DNA (usually
  fewer than 3000 base pairs) to be amplified to
  about a million fold
• This amplified sample then allows for size
  determination and nucleotide sequencing
• Introduced in 1985 by Kary Mullis
• Millions of copies of a segment of DNA can be
  made within a few hours.

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Three Steps

• Separation Double Stranded DNA is denatured by
  heat into single strands.
• Short Primers for DNA replication are added to
  the mixture.
• DNA polymerase catalyzes the production of
  complementary new strands.
• Copying The process is repeated for each new
  strand created
• All three steps are carried out in the same vial
  but at different temperatures

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Step 1 Separation

• Combine Target Sequence, DNA primers template,
  dNTPs, TAQ Polymerase
• Target Sequence Usually fewer than 3000 bp
• Identified by a specific pair of DNA primers-
  usually oligonucleotides that are about 20
  nucleotides
• Heat to 95 degrees Celsius to separate strands
  (for 0.5-2 minutes)
• Longer times increase denaturation but decrease
  enzyme and template

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Magnesium as a Cofactor

• Stabilizes the reaction between
• oligonucleotides and template DNA
• DNA Polymerase and template DNA

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• Heat Denatures DNA by uncoiling the Double Helix
  strands.

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Step 2 Priming

• Decrease temperature by 15-25 degrees
• Primers anneal to the end of the strand
• 0.5-2 minutes
• Shorter time increases specificity but decreases
  yield
• Requires knowledge of the base sequences of the
  3 - end

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Selecting a Primer

• Primer length
• Melting Temperature (Tm)
• Specificity
• Complementary Primer Sequences
• G/C content and Polypyrimidine (T, C) or
  polypurine (A, G) stretches
• 3-end Sequence
• Single-stranded DNA

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Step 3 Polymerization

• Since the Taq polymerase works best at around 75
  degrees C (the temperature of the hot springs
  where the bacterium was discovered), the
  temperature of the vial is raised to 72-75
  Degrees Celsius
• The DNA polymerase recognizes the primer and
  makes a complementary copy of the template which
  is now single stranded.
• Approximately 150 nucleotides/sec

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Potential Problems with Taq

• Lack of proof-reading of newly synthesized DNA.
• Potentially can include diNucleotriphosphates
  (dNTPs) that are not complementary to the
  original strand.
• Errors in coding result
• Recently discovered thermostable DNA polymerases,
  Tli and Pfu, are less efficient, yet highly
  accurate.

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Amplification
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PCR Applications

• Detection of infectious diseases
• Detection of variations and mutations in genes
• Detection of diseases from the past
• PCR and the law

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Detection of infectious diseases

• - AIDS Virus
• - Otitis Media-middle ear infection
• - Lyme Disease-joint inflammation from tick
  bites
• - Detect 3 sexually transmitted diseases in one
  swab-herpes, papillomarvirus, chlamydia
•  -Test to see if mother and baby have compatible
  blood group-saves lives of babies

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Detection of Variations and Mutations in Genes

• Detects people with inherited disorders
• Lets us know who carries deleterious variations
  (mutations)
• Direct way of distinguishing among the confusion
  of different mutations in a single gene. Ex
  Duchenne muscular dystrophy
• Track presence or absence of DNA abnormalities
  characteristic to cancer

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Detection of diseases from the past

• Presidential candidate Humphreys-had cancer
• John Dalton-was colored blind and realized that
  this was the case because he lacked a gene for
  one of the three photopigments, which caused him
  to be color blind

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PCR and the Law

• DNA fingerprinting
• Can multiply small amounts of DNA found in blood
  samples, hair, semen, and other body fluids
• Proving innocence of those already convicted
• Kirk Bloodsworth-wrongly accused of raping and
  murdering a nine year old. Using PCR, he was
  proved innocent and released from prison in 1993

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Future of PCR

• Copying larger pieces of DNA
• Miniaturization of hardware (chip-sized devices)
• Computer automated test and analysis
• Taking PCR on the road and getting on the spot
  DNA analysis
• Diagnose infection or genetic disorder right in
  the doctors office

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References

• Polymerase Chain Reaction-Xeroxing DNA
  http//www.accessexcellence.org/AB/IE/PCR_Xeroxing
  _DNA.html
• The Polymerase Chain Reaction
  http//avery.rutgers.edu/WSSP/StudentScholars/proj
  ect/archives/onions/pcr.html
• Polymerase Chain reaction http//www.tulane.edu/
  wiser/methods/handouts/pcr.PDF
• Diagrams from http//allserv.rug.ac.be/avierstr
  /principles/pcrani.html
• Purves, Sadava, Orians, Heller. Life. 6th ed.
  Sinauer Associates, 2001.
• Mechanism of PCR. http//usitweb.shef.ac.uk/mb
  a97cmh/tutorial/pcr.htm
• The polymerase Chain Reactionwww.faseb.org/opar/
  bloodsupply/pcr.html