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PCR3

PCR is used to amplify specific DNA fragments. It involves repeated cycles of heating and cooling of the DNA sample to denature, anneal primers, and extend new strands. The components required are template DNA, primers, DNA polymerase, dNTPs, and buffer. Key variables include temperatures, cycle times, primer design, and choice of polymerase. With repeated cycling, specific DNA fragments can be exponentially amplified over a million-fold from tiny starting quantities.

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124 views

PCR3

PCR is used to amplify specific DNA fragments. It involves repeated cycles of heating and cooling of the DNA sample to denature, anneal primers, and extend new strands. The components required are template DNA, primers, DNA polymerase, dNTPs, and buffer. Key variables include temperatures, cycle times, primer design, and choice of polymerase. With repeated cycling, specific DNA fragments can be exponentially amplified over a million-fold from tiny starting quantities.

Uploaded by

vivitri.dewi
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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PCR Basics

1. Purpose of PCR
2. Overview
3. Components of PCR Reaction
4. Variables
• Temperature
• Cycle Times and Numbers
• Primer
• Buffer
• Polymerase
5. Experimental Notes
Polymerase Chain Reaction
• “Amplify” large quantities of DNA (μg quantities)
from small quantities (ag quantities) [Trillion fold
amplification]

• Analyze single DNA fragments out of large


complex mixture. [ Human genome mixture of 12
million 300bp fragments]

• Alter DNA sequence – directed mutagenesis.


Overview of PCR
1. Temperature Cycling

Denaturation 94°
Annealing 55°
Extension 72°

2. Every Cycle DNA


between primers is
duplicated
PCR Amplification
Exponential Amplification

30 cycles --- 2 Trillion copies in theory


Components of PCR Reaction
• Template DNA
• Flanking Primers
• Thermo-stable polymerase
– Taq Polymerase Thermus aquaticus

• dNTP
– (dATP, dTTP, dCTP, dGTP)
• PCR Buffer (mg++)
• Thermocyler
PCR Variables
1. Temperature
2. Cycle Times and Temps
3. Primer
4. Buffer
5. Polymerase
Temperature
• Denaturation
– Trade off between denaturing DNA and not denaturing Taq Polymerase
• Taq half-life 40min at 95 °, 10min at 97.5°
– 95°

• Annealing
– Trade off between efficient annealling and specificity
– 2-5 ° below Tm

• Extension
– Temperature optimum for Taq
Polymerase
– 72 °
Cycle Times and Temps
Typical PCR Run
Step Time/Temp
1 3 min at 95°
2 30 sec at 95°
3 1 min at 55°
4 2 min at 72°
5 Go to step 2 - 29 times
6 8 min 72°
7 0 min 4°
8 End
Primers
• Paired flanking primers
• Length (17-28bp)
• GC content 50-60%
• GC Clamp
• Tm’s between 55-80
• Avoid simple sequences – e.g. strings of G’s
• Avoid primer self complementary
– e.g. hairpins, homodimers, heterodimers
PCR Buffer
• Basic Components
– 20mM Tris-HCL pH 8.4
– 50mM KCl
– 1.5 mM MgCl2
• Magnesium – Since Mg ions form complexes with dNTPs, primers and
DNA templates, the optimal concentration of MgCl2 has to be selected
for each experiment. Too few Mg2+ ions result in a low yield of PCR
product, and too many increase the yield of non-specific products and
promote misincorporation.
• Potential Additives
– Helix Destabilisers - useful when target DNA is high G/CWith NAs of high
(G+C) content.
• dimethyl sulphoxide (DMSO),
• dimethyl formamide (DMF),
• urea
• formamide
– Long Targets >1kb. Formamide and glycerol
– Low concentration of template: Polyethylene glycol (PEG)
PCR Polymerases
• Taq, Vent, Pfu, others
• Native or Cloned
• Half-life
– e.g. Taq 40 min half-life, Vent 7 hour half-life
• 3’-5’ Exo nuclease – proofreading
• Fidelity (Error Rate
– Taq 1/10,000nt, Pfu 1/1,000,000
• Processivity
• Extra bases at end
Notes
• Typical Reaction
• 80 μl dH2O
• 10 μl 10 X PCR buffer + mg
• 2 μl 200 μM dNTP
• 1 μl 50 μM Left Primer
• 1 μl 50 μM Right Primer
• 5 μl Worm Lysate
• 1 μl Taq Pol (5 Units/ μl)
100 μl Total Vol
Master Mix
• 1 Reaction Common Comp
– 80 μl dH2O
– 10 μl 10 X PCR buffer
– 2 μl 200 μM dNTP
– 1 μl Taq Pol (5 Units/ μl)
93 μl Total Volume

• 9.5 reaction master mix – add 93 μl to each reaction


– 760 μl dH2O
– 95 μl 10 X PCR buffer
– 19 μl 200 μM dNTP
– 9.5 μl Taq Pol (5 Units/ μl)

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