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P olymerase C hain R eaction Enzyme that replicates DNA or RNA A reaction that, once started, uses its own product as a reactant… thus the reaction feeds itself leading to exponential growth How does PCR work? Start ( 1 copy ) 1. Melting (~94 °, 30 sec)

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p olymerase c hain r eaction
Polymerase Chain Reaction

Enzyme that replicates DNA or RNA

A reaction that, once started, uses its own product as a reactant… thus the reaction feeds itself leading to exponential growth

how does pcr work
How does PCR work?

Start (1 copy)

1. Melting (~94°, 30 sec)

2. Primer Annealing (~45°- 65°, 30 sec)

3. Primer Extension (~72°, 1 min per kb)

Finish (2 copies)

exponential amplification
Exponential amplification

Start – 1 copy

4th Cycle – 16 copies

10th Cycle – 1024 copies

20th Cycle – 1.05 million copies

30th Cycle – 1.07 billion copies

# of copies = 2n, (n = # of cycles)

1st Cycle – 2 copies

2nd Cycle – 4 copies

3rd Cycle – 8 copies

what is pcr used for
What is PCR used for?
  • The short answer: just about anything with DNA (or RNA)
  • Cloning into plasmids, etc.
  • cDNA creation
  • Mutagenesis
  • DNA (or RNA) quantification
  • Radioactive and non-radioactive labels
  • Screening bacterial clones for a correct plasmid
  • Genotyping
  • DNA sequencing
pcr methods
PCR Methods
  • Conventional PCR
  • Reverse-transcriptase (RT) PCR
  • Real-time PCR
ingredients
Ingredients
  • ReactantsFinal concentration
  • PCR reaction buffer (10X) … dilute 1:10
  • Magnesium (MgCl2 or MgSO4) ~1-5 mM (start with 2 mM)
  • dNTPs 0.2 mM each
  • Upstream primer ~0.5 mM
  • Downstream primer ~0.5 mM
  • Polymerase ~2.5 Units*
  • Template DNA 20-750 ng**
  • Mineral oil overlay?

*follow instructions that come with polymerase

**for plasmid DNA, use ~20 ng per 100uL reaction

**for genomic DNA, use ~100-750 ng per 100uL reaction

tweaking the reaction
Tweaking the reaction
  • Magnesium
    • Affects the binding of primers to the template DNA and the efficiency of the reaction
    • If you get little or no product, try repeating the reaction with a range of [Mg]
  • Template DNA
    • The purity of the template DNA will affect the reaction
    • Too much DNA can inhibit the reaction… sometimes less is more!
  • Primer Annealing temperature
    • Start out with this set a couple degrees lower than the melting temperature of your primers and adjust if necessary.
    • If you don’t get any product, try lowering the annealing temperature
    • If you get non-specific bands, try raising the annealing temperature
polymerases
Polymerases

Taq: old faithful, the original PCR polymerase now comes in every possible flavor (including Patton taq)

High-fidelity: stays on the DNA better and has proofreading activity, so it amplifies longer templates with less errors (up to 12 kb) [examples: PWO, Taq HiFi, Pfu]

“Hotstart”: polymerase is inactive until heated to 94°… this helps to reduce non-specific amplification (improvise this by not adding the polymerase until the first cycle has reached 94°)

primers
Primers
  • How long do you need them to be?
    • 4n = chance that you will get a random match (n = length of primer)
    • 15 nt long = 1 match per ~1 billion base pairs
    • 20 nt long = 1 match per ~1 trillion base pairs (human genome is 3 billion)
  • Watch for primer homology and possible secondary structures
    • Primer pairs should not complement each other, especially at the 3’ end
    • Stretches of 5 G’s will form a secondary structure… also avoid repetitive sequences
  • Make sure you design the primers in the right orientation!!!
designing primers

A/T = 10, G/C = 10

Tm = 55°

A/T = 10, G/C = 10

Tm = 55°

Designing primers

***Melting temperature shortcut:

G or C = 4°

A or T = 2°

  • Count the # of A/T and G/C in your primer
  • Tm = 2*(A+T) + 4*(G+C) - 5°
  • So, a 20 bp primer with 50% GC will have a Tm of 55° [2*10 +4*10 – 5 = 55]

5’-ACGATGCATGTAGCTACGTAGG…[YFG]…CGGGTACTGAACGGCAAACCTAGTC-3’

3’-TGCTACGTACATCGATGCATCC….[YFG]…GCCCATGACTTGCCGTTTGGATCAG-5’

controls
Controls
  • Negative Control (VERY IMPORTANT)
    • Add water instead of template DNA
    • Confirms that your reagents are not contaminated
  • Positive Control
    • If possible, run a parallel reaction with something that you know the primers will amplify
slide12

Experiment:

  • The DHFR gene is present in hamster CHOK-1 cells. You would like to put the hamster DHFR gene in human Hela cells and confirm integration by PCR.
  • You don’t think your primers will amplify a target from Hela cells, but you’d like to be sure.
  • You have the DHFR gene from CHOK-1 cloned onto a plasmid.

DHFR (plasmid)

CHOK-1 DNA

Hela DNA

Water (no DNA)

Marker 1

Marker 2

case 1
Case 1

DHFR (plasmid)

CHOK-1 DNA

Hela DNA

Water (no DNA)

Marker 1

Marker 2

  • Problem: No amplification
  • Positive control didn’t amplify, so reaction conditions are bad
  • Solutions?
  • Re-run reaction… maybe you forgot to add something
  • Lower primer annealing temperature
  • Check primer design
  • Try different Mg concentrations
case 2
Case 2

DHFR (plasmid)

CHOK-1 DNA

Hela DNA

Water (no DNA)

Marker 1

Marker 2

  • Problem: Negative control has a band
  • Your reagents may be contaminated
  • Solutions?
  • Re-do the reaction… maybe you dropped a skin flake in the reaction mix or something
  • Discard old solutions and primers and use fresh materials
  • Set up the reaction away from where you usually do experiments… DNA around your work area can end up in your reactions
slide15

Case 3: screening clones

Positive Control

Clone #1

Clone #2

Clone #3

Clone #4

Clone #5

Water (no DNA)

Marker

  • Problem: Too many bands
  • Your template DNA may be contaminated
  • Your primers may not be specific
  • Solutions?
  • Raise the annealing temperature to make your primers more specific
  • Try altering your primer design if possible
  • Try using a different sample of template DNA if possible
general tips
General tips
  • Set up your PCR reactions away from where you usually work
  • Use sterile technique and wear gloves
  • Filtered tips aren’t a bad idea
  • Design primers with similar melting temperatures
  • Add your template DNA last
pcr applications radiolabelled probe
PCR applications: radiolabelled probe
  • For Southerns, Northerns, etc.

Probe sequence

primers

  • Replace dATP with 32P dATP or use radiolabelled primers
  • Run PCR using normal conditions

Radioactive probe complementary to your target sequence

pcr applications dna quantitation
PCR applications: DNA quantitation
  • Conventional PCR
  • Semi-quantitative at best…
  • You only measure the end product, which only gives you a rough idea of the amount of starting template
  • Real-time PCR
  • quantitative
  • Measures the production of dsDNA as it is made using a fluorescent dye, so you can monitor the reaction in “real-time” and accurately measure the amount of starting template
core facilities
Core facilities
  • 740 Light Hall: get everything you need for PCR right here, including primers and kits
  • Real-time PCR thermocycler:
got questions
Got Questions?
  • Steven Gray
    • Ellen Fanning’s lab, 2325 Stevenson Center (on the far side of MRBIII, on the 3rd floor)
    • 343-5802
    • Steven.j.gray@vanderbilt.edu