RAPD PCR

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RAPD PCR

• Douglas J. Burks
• Department of Biology
• Wilmington College of Ohio

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PCR

• What is PCR
• What is PCR 2
• Virtual lab

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PCR

• The Polymerase Chain Reaction
• Uses DNA replication to amplify a sequence
• DNA polymerase
• Needs a primer
• Nucleotides
• Template strand
• Goes only in 5 ? 3 direction

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PCR

• Use to detect if a specific DNA is present in a
  sample
• Primers give selectivity of sequence replicated.
• Need separate primers for each strand

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RAPD

• How It Works
• Unlike traditional PCR analysis, RAPD (pronounced
  "rapid") does not require any specific knowledge
  of the DNA sequence of the target organism the
  identical 10-mer primers will or will not amplify
  a segment of DNA, depending on positions that are
  complementary to the primers' sequence. For
  example, no fragment is produced if primers
  annealed too far apart or 3' ends of the primers
  are not facing each other. Therefore, if a
  mutation has occurred in the template DNA at the
  site that was previously complementary to the
  primer, a PCR product will not be produced,
  resulting in a different pattern of amplified DNA
  segments on the gel.

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RAPDs

• Use a random sequence as a primer
• Tenmer
• 5GGCCATTGAG3
• Occurrence per genome
• Definitions
• E(d) number of primer sites per genome
• G genome complexity
• k the size of the primer
• d the distance between the inverted repeat units
  ie.
• P probability of getting a specific base (0.25)
• E(d) g x pk
• 2 x 109 x 0.2510
• 1907 times in human genome

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RAPDs

• Not really random
• Primer sequences occur at specific locations
• Researcher does not know where
• Mutation in primer will lead to loss of bands
• Mutation in region to create a new primer
  sequence will add bands

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10X Qiagen Buffer. Contains TrisCl, KCl,
(NH4)2SO4, 15 mM MgCl2 pH 8.7 (20C)
Master Mix Pipetting common elements needed for
several reactions
Use 2 uL of DNA 20 ng but dont know for
sure. 48 uL master mix Thermocycler Profile 94oC
30 seconds 36oC 30 seconds 72oC 1
minute 33 cycles 1 cycle at 72oC for 4
min Cool at 4oC