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Randomly
Amplified
Polymorphic
DNA
• Genetic markers resulting from PCR amplification of genomic DNA
segments recognized by random primers of arbitrary nucleotide
sequence.
• primers usually 10 mers
• GC rich.
• annealing temperature: low 35° C.
• amplified products are detected in agarose gels
PCR
• Buffer (containing Mg++)
• Template DNA
• 2 Primers that flank the fragment
of DNA to be Amplified
• dNTPs
• Taq DNA Polymerase (or another
thermally stable DNA polymerase)
RAPD
• Buffer (containing Mg++) usually
high Mg++ concentrations are used
lowering annealing stringency
• Template DNA
• 1 short primer (10 bases)not
known to anneal to any specific
part of the template DNA
• dNTPs
• Taq DNA Polymerase (or another
thermally stable DNA polymerase)
Advantageous
• Primer sequences are synthesized at
random (does not require any
sequences information).
• No gel transfer or hybridization is
required.
• Fast (many of the steps are
automated ).
• Relatively inexpensive
• Highly variable.
• The amount of template DNA is
extremely small.
Disadvantages
• Markers are dominant ( Presence of a
band could mean the individual is
either heterozygous or homozygous
for the sequence--can’t tell which ).

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RAPDs (Randomly Amplified Polymorphic DNA).pdf

  • 2. • Genetic markers resulting from PCR amplification of genomic DNA segments recognized by random primers of arbitrary nucleotide sequence. • primers usually 10 mers • GC rich. • annealing temperature: low 35° C. • amplified products are detected in agarose gels
  • 3. PCR • Buffer (containing Mg++) • Template DNA • 2 Primers that flank the fragment of DNA to be Amplified • dNTPs • Taq DNA Polymerase (or another thermally stable DNA polymerase) RAPD • Buffer (containing Mg++) usually high Mg++ concentrations are used lowering annealing stringency • Template DNA • 1 short primer (10 bases)not known to anneal to any specific part of the template DNA • dNTPs • Taq DNA Polymerase (or another thermally stable DNA polymerase)
  • 4.
  • 5.
  • 6. Advantageous • Primer sequences are synthesized at random (does not require any sequences information). • No gel transfer or hybridization is required. • Fast (many of the steps are automated ). • Relatively inexpensive • Highly variable. • The amount of template DNA is extremely small. Disadvantages • Markers are dominant ( Presence of a band could mean the individual is either heterozygous or homozygous for the sequence--can’t tell which ).