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Romans 5:17

Romans 5:17 17 For if by one man’s offence death reigned by one; much more they which receive abundance of grace and of the gift of righteousness shall reign in life by one, Jesus Christ. Random Amplified Polymorphic DNA RAPD. Timothy G. Standish, Ph. D. History.

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Romans 5:17

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  1. Romans 5:17 17 For if by one man’s offence death reigned by one; much more they which receive abundance of grace and of the gift of righteousness shall reign in life by one, Jesus Christ.

  2. Random Amplified Polymorphic DNARAPD Timothy G. Standish, Ph. D.

  3. History • Shortly after Kary Mullis invented the Polymerase Chain Reaction (PCR) it was realized that short primers would bind to several locations in a genome and thus could produce multiple fragments • Williams et al. (1990) developed Random Amplified Polymorphic DNA (RAPD) a technique using very short 10 base primers to generate random fragments from template DNAs • RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprint • Techniques related to RAPD include: • DNA Amplification Fingerprinting (DAF) - Caetano-Anolles et al. (1991) uses very short (eight nucleotide long) primers • Arbitrary Primed PCR (AP-PCR) - Welsh and McClelland (1990) uses longer primers, but lowers primer annealing stringency to get priming at many sites

  4. Components of a PCR and RAPD Reactions 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) 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)

  5. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers 72 oC Temperature 50 50 oC 0 T i m e 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’

  6. PCR Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ 5’ 3’

  7. PCR Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ Heat 5’ 3’

  8. PCR Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’

  9. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’ Heat Heat 5’

  10. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’

  11. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ 5’ Heat Heat

  12. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’

  13. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ Fragments of defined length 5’ 5’ 5’ 5’ 5’

  14. DNA Between The Primers Doubles With Each Thermal Cycle Number 1 2 4 8 16 32 64 0 Cycles 1 2 3 4 5 6

  15. Modifying Thermal Cycling • Two modifications made to typical thermal cycling when RAPD is being done: • Annealing temperatures are generally very low, around 36 oC - This allows very short primers to anneal to template DNA • More thermal cycles are used, typically 45 - This compensates for the inefficiency which results from using such short primers.

  16. RAPD Template DNA • Primer binds to many locations on the template DNA • Only when primer binding sites are close and oriented in opposite direction so the primers point toward each other will amplification take place

  17. RAPD Template DNA Primers point away from each other, so amplification won’t happen

  18. RAPD Template DNA Primers point in the same direction, so amplification won’t happen

  19. RAPD Template DNA > 2,000 bases Primers too far apart, so amplification won’t happen

  20. RAPD Template DNA Primers are just the right distance apart, so fragment is amplified 100 - 1,500 bases

  21. Separated RAPD Fragments 4mM MgCl2 1.2 U Taq 5 pM OPA-16 4mM MgCl2 0.6 U Taq 10 pM OPA-16 2mM MgCl2 1.2 U Taq 10 pM OPA-16 M M 2 3 4 5 6 7 8 9 10 RAPD reactions were run in groups of 3 using the same template and primer, but varying Magnesium, polymerase and primer concentrations Which variable has the greatest impact on fragment patterns? Lowering Magnesium ion concentration results in loss of the largest fragment visible in lanes 2-7 Normal concentrations are shown in yellow text. M = A size standard

  22. The End

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