©2001 timothy g. standish romans 5:17 17for if by one man’s offence death reigned by one; much...
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©2001 Timothy G. Standish
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.
©2001 Timothy G. Standish
Random Amplified Random Amplified Polymorphic DNAPolymorphic DNA
RAPDRAPDTimothy G. Standish, Ph. D.
©2001 Timothy G. Standish
HistoryHistory 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
©2001 Timothy G. Standish
Components of a PCR and Components of a PCR and RAPD ReactionsRAPD Reactions
RAPD1. 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)
PCR1. Buffer (containing Mg++)
2. Template DNA3. 2 Primers that flank the
fragment of DNA to be amplified
4. dNTPs5. Taq DNA Polymerase (or
another thermally stable DNA polymerase)
©2001 Timothy G. Standish
PCRPCRMelting
94 oC
Melting
94 oC
AnnealingPrimers
50 oC
Extension
72 oCT
empe
ratu
re
100
0
50
T i m e
30x
5’3’
3’5’
3’5’
5’
5’3’5’
3’5’
5’
5’
5’
5’3’
3’5’
3’5’
5’3’
5’3’
5’
©2001 Timothy G. Standish
PCRPCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
5’3’
3’5’
©2001 Timothy G. Standish
PCRPCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’
Heat
©2001 Timothy G. Standish
PCRPCRMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’5’
5’
Melting94 oC
©2001 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
©2001 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
©2001 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
Heat
Heat
©2001 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
©2001 Timothy G. Standish
Fragments of defined length
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’ 5’
5’
5’
5’
5’
5’
5’
5’
©2001 Timothy G. Standish
DNA Between The Primers Doubles DNA Between The Primers Doubles With Each Thermal CycleWith Each Thermal Cycle
0Cycles
Number1
3
8
2
4
1
2
4
16
5
32
6
64
©2001 Timothy G. Standish
Modifying Thermal CyclingModifying Thermal Cycling Two modifications made to typical thermal
cycling when RAPD is being done:
1. Annealing temperatures are generally very low, around 36 oC - 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.
©2001 Timothy G. Standish
RAPDRAPD
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
©2001 Timothy G. Standish
RAPDRAPD
Template DNA
Primers point away from each other, so amplification won’t happen
©2001 Timothy G. Standish
RAPDRAPD
Template DNA
Primers point in the same direction, so amplification won’t happen
©2001 Timothy G. Standish
RAPDRAPD
Template DNA
Primers too far apart, so amplification won’t happen
> 2,000 bases
©2001 Timothy G. Standish
Template DNA
Primers are just the right
distance apart, so fragment is
amplified
100 - 1,500 bases
RAPDRAPD
©2001 Timothy G. Standish
MM 2 3 4 5 6 7 8 9 10
Separated RAPD FragmentsSeparated RAPD Fragments4mM MgCl2
1.2 U Taq5 pM OPA-16
4mM MgCl2
0.6 U Taq10 pM OPA-16
2mM MgCl2
1.2 U Taq10 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?
©2001 Timothy G. Standish