1. ©2001 Timothy G. Standish Romans 5:17 17For 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. ©2001 Timothy G. Standish Random Amplified Polymorphic DNA RAPD Timothy G. Standish, Ph. D.

3. ©2001 Timothy G. Standish 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. ©2001 Timothy G. Standish Components of a PCR and RAPD Reactions RAPD 1. Buffer (containing Mg ++ ) - usually high Mg ++ concentrations are used lowering annealing stringency 2. Template DNA 3. 1 short primer (10 bases) not known to anneal to any specific part of the template DNA 4. dNTPs 5. Taq DNA Polymerase (or another thermally stable DNA polymerase) PCR 1. Buffer (containing Mg ++ ) 2. Template DNA 3. 2 Primers that flank the fragment of DNA to be amplified 4. dNTPs 5. Taq DNA Polymerase (or another thermally stable DNA polymerase)

5. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 5’3’ 5’ 3’5’ 3’ 5’ 3’5’ 3’ 5’ 3’5’ 3’ 5’3’ 5’

6. ©2001 Timothy G. StandishPCR Melting 94 o C Temperature 100 0 50 T i m e 5’3’ 5’

7. ©2001 Timothy G. StandishPCR Melting 94 o C Temperature 100 0 50 T i m e 3’5’ 3’ Heat

8. ©2001 Timothy G. StandishPCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 3’5’ 3’ 5’ Melting 94 o C

9. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ Heat 5’

10. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ 5’

11. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ 5’ Heat

12. ©2001 Timothy G. StandishPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ 5’

13. ©2001 Timothy G. Standish Fragments of defined lengthPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 3’5’ 3’ 5’

14. ©2001 Timothy G. Standish DNA Between The Primers Doubles With Each Thermal Cycle 0 Cycles Number 1 3 8 2 4 1 2 4 16 5 32 6 64

15. ©2001 Timothy G. Standish Modifying Thermal Cycling Two modifications made to typical thermal cycling when RAPD is being done: 1. Annealing temperatures are generally very low, around 36 o C - This allows very short primers to anneal to template DNA 2. More thermal cycles are used, typically 45 - This compensates for the inefficiency which results from using such short primers.

16. ©2001 Timothy G. StandishRAPD 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. ©2001 Timothy G. StandishRAPD Template DNA Primers point away from each other, so amplification won’t happen

18. ©2001 Timothy G. StandishRAPD Template DNA Primers point in the same direction, so amplification won’t happen

19. ©2001 Timothy G. StandishRAPD Template DNA Primers too far apart, so amplification won’t happen > 2,000 bases

20. ©2001 Timothy G. Standish Template DNA Primers are just the right distance apart, so fragment is amplified 100 - 1,500 basesRAPD

21. ©2001 Timothy G. Standish MM 2 3 4 5 6 7 8 9 10 Separated RAPD Fragments 4mM MgCl 2 1.2 U Taq 5 pM OPA-16 4mM MgCl 2 0.6 U Taq 10 pM OPA-16 2mM MgCl 2 1.2 U Taq 10 pM OPA-16 Normal concentrations are shown in yellow text. M = A size standard Lowering Magnesium ion concentration results in loss of the largest fragment visible in lanes 2-7 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?

22. ©2001 Timothy G. Standish