PCR History (PowerPoint) by yurtgc548

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									Advanced PCR
Dave Palmer, Byotix, Inc.
Advanced PCR
 PCR of Plant Material
 Multiplex PCR
 Modifications to Standard PCR
 PCR Troubleshooting
        Plant DNA Extraction
                 Rapid 3-Step
                 Extraction Method

                 Freeze
                 TPS
                      100 mM Tris pH 9.5
                      1 M KCl
                      10 mM EDTA
                 Heat 95°C

D. Thomson and R. Henry, 1995, Single-step protocol for preparation of plant tissue for analysis by PCR, BioTechniques, 19:394-400
How PCR works
 Cold Spring Harbor Animation



PCR.EXE
Review: The structure of DNA




  Helix       Complementary Base Pairing
Multiplex PCR: What
PCR using several
primer pairs
SIMULTANEOUSLY
Typically generates a
product band for each
primer pair
Multiplex PCR: Why
Detect several genes at
once
   eg. transgenic plant screen
Internal controls
   VERY important
   Tells you how well the PCR
    reaction worked
   Reduces “false negatives”
Multiplex PCR: How
Same as regular PCR
Care in primer design
   Much greater chance of
    primer-dimers
   Annealing temperatures
    must be close
   Much greater chance of
    artifacts
A Typical PCR Reaction
   Component            ml
Sterile Water          38.0
10X PCR Buffer          5.0
MgCl2 (50mM)            2.5
dNTP’s (10mM each)      1.0
 PrimerFWD (25 pmol/ul) 1.0
 PrimerREV              1.0
DNA Polymerase          0.5
DNA Template            1.0

Total Volume          50.0
A Typical Multiplex PCR Reaction
   Component           ml
 Sterile Water        34.0
 10X PCR Buffer        5.0
 MgCl2 (50mM)          2.5
 dNTP’s (10mM each)    1.0
  Primer1FWD           1.0
  Primer1REV           1.0
  Primer2FWD           1.0
  Primer2REV           1.0
  Primer3FWD           1.0
  Primer3REV           1.0
 DNA Polymerase        0.5
 DNA Template          1.0

 Total Volume         50.0
Multiplex PCR: Example
                Three primer pairs
                   Effect, Marker, and
                    Internal Control genes
 Effect Gene
                Control is smallest
 Marker Gene
 Control Gene
                fragment, also brightest
                band
                Effect is largest
                fragment, faintest band
Multiplex PCR: Example
 Three primer pairs

 Effect Gene
 Marker Gene
 Control Gene

                      Which are transgenic?
  Multiplex PCR
                  Problem:
                     If the control gene product is
                      the BRIGHTEST in a set, then
                      it’s very difficult to tell the
Effect Gene?          difference between weak PCR
Marker Gene?          reactions and nontransgenics.
Control Gene
                  Solution:
                     Redesign primers.
Multiplex PCR: Example
                Three primer pairs
 Control Gene
                   Control, marker, and
 Marker Gene
                    effect genes
 Effect Gene    Control gene fragment
                is largest and (almost)
 Primers
                faintest
                Effect gene is smallest
                and brightest
Multiplex PCR: Example
 Three primer pairs

 Control Gene
 Marker Gene
 Effect Gene

 Primers
                      Which are transgenic?
Other Types of PCR
  Different templates
     Nested PCR
     RT-PCR
  Unusual protocols
     iPCR
     Real-time PCR
PCR Troubleshooting
The effect of each component
PCR Reaction Components
  Water
  Buffer
  DNA template
  Primers
  Nucleotides
  Mg++ ions
  DNA Polymerase
  Extras
PCR Reaction Components
  Water        Purity
               Contamination
                   Amplification
                    Products
PCR Reaction Components
  Buffer   Must match polymerase
           Typically contain KCl and Tris
           Can vary over a slight range:
              Not much difference in range
               from 0.8 X to 2.0 X
              Primer efficiency reduced
               outside this range




                     http://info.med.yale.edu/genetics/ward/tavi/p06.html
PCR Reaction Components
DNA template   Amount of DNA present
                  Less DNA means more cycles
               Complexity of DNA
                  Eg. plasmid vs. whole genome
               Purity
                  Interfering factors, eg. enzymes, salts
               Degradation
                  PCR more forgiving of degraded DNA
               Contamination
                  Amplification products
               Presence of “poisons”
                  Eg. EDTA which scavenges Mg++
PCR Reaction Components
  Primers   Age
            Number of freeze-thaws
            Contamination
            Amount
               Can vary over a wide range (50X)
               100-500 nM typical
               Too low: low amplification
               Too high: low amplification

                        http://info.med.yale.edu/genetics/ward/tavi/p05.html
PCR Reaction Components
Nucleotides   20-400 uM works well
                 Too much: can lead to mispriming and
                  errors
                 Too much: can scavenge Mg++
                 Too low: faint products
              Age
              Number of freeze-thaws
                 Just 3-5 cycles is enough to make PCRs
                  not work well
              Dilute in buffer (eg. 10mM Tris pH
              8.0 to prevent acid hydrolysis)
              Contamination

                           http://info.med.yale.edu/genetics/ward/tavi/p13.html
PCR Reaction Components
Mg++ ions   Mg is an essential cofactor
            of DNA polymerase
            Amount can vary
               0.5 to 3.5 uM suggested
               Too low: Taq won’t work
               Too high: mispriming




                     http://info.med.yale.edu/genetics/ward/tavi/p14.html
PCR Reaction Components
  Bottom Line:
     All components work over a wide range.
     Need to avoid contamination.
     Optimization by trial-and-error.
PCR Reaction Components
DNA Polymerase   Thermostable?
                    Activity declines with time at
                     95C
                 Matches buffer?
                 Age
                 Contamination
                 Concentration: Typically 0.5
                 to 1.0 U/rxn

                           http://info.med.yale.edu/genetics/ward/tavi/p12.html
PCR Reaction Components
Extras   Proprietary or added by user
         Glycerol, DMSO
            Stabilize Taq, decrease secondary
             structure
            May help or hurt, depending on primers
            Typically already in the Taq stock
         BSA
            Frequently helps, doesn’t hurt
         Betaine
            Useful for GC-rich templates


                          http://info.med.yale.edu/genetics/ward/tavi/p16.html
                          http://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html
PCR Cycling Parameters
  Denaturation Temp
  Annealing Temp
  Extension Temp
  Time
  Number of Cycles
  Reaction Volume
  “Odd” Protocols
PCR Cycling Parameters
Denaturation Step   Must balance DNA
                    denaturation with Taq
                    damage
                    95C for 30 - 60s typically is
                    enough to denature DNA
                    Taq loses activity at high
                    temps:
                       Half-life at 95C: 40 min
                       Half-life at 97.5C: 5 min


                           http://info.med.yale.edu/genetics/ward/tavi/p08.html
PCR Cycling Parameters
Annealing Step   Most critical step
                 Calculate based on Tm
                    Often does not give expected results
                 Trial-and-Error
                    Almost always must be done anyway
                    Too hot: no products
                    Too cool: non-specific products
                 Gradient thermocyclers very useful
                 Typically only 20s needed for
                 primers to anneal

                              http://info.med.yale.edu/genetics/ward/tavi/p08.html
PCR Cycling Parameters
  Extension Step   Temperature typically 72C
                      Reaction will also work well
                       at 65C or other temps
                   Time (in minutes) roughly
                   equal to size of the largest
                   product in kb
                      Polymerase runs at 60bp/s
                       under optimum conditions
                   Final “long” extension step
                   unnecessary

                             http://info.med.yale.edu/genetics/ward/tavi/p08.html
                             http://info.med.yale.edu/genetics/ward/tavi/p10.html
PCR Cycling Parameters
Number of Cycles   Number of source molecules:
                      >100,000: 25-30
                      >10,000: 30-35
                      >1,000: 35-40
                      <50: 20-30 fb. nested PCR
                   Do not run more than 40
                      Virtually no gain
                      Extremely high chance of non-
                       specific products
                   Best optimized by trial-and-
                   error


                           http://info.med.yale.edu/genetics/ward/tavi/p08.html
PCR Cycling Parameters
Reaction Volume   Doesn’t affect PCR results
                  as long as volume is within
                  limits.
                  Heated lid important.
                  5ul, 20ul, 100ul all work.
                  Slightly higher yield with
                  lower volumes.


                       http://info.med.yale.edu/genetics/ward/tavi/p03.html
PCR Cycling Parameters
“Odd” Protocols   Hot-Start PCR
                     Taq is added last
                  Touchdown PCR
                     Annealing temp is
                      progressively reduced
Adventures in PCR
There’s a fly in my primer!
The protocol that never worked.
“There’s A Fly In My Primer!”
This happened when we were
first developing PCR methods.
Transgene-specific primers not
available for testing.
Needed primers to test DNA
extraction protocol, multiplexing.
Synthesized “NS”-series primers
   Pastrik, 2000
   A “plant-specific primer set”
    suitable for potato amplification.
“There’s A Fly In My Primer!”
First Experiment:
   DNA extraction and multiplexing
   Worked great!
Second Experiment
   To confirm first results
   Great, but “ghost” bands in
    controls. No cause for alarm.
   Implemented contamination
    controls.
“There’s A Fly In My Primer!”
Third Experiment
   To determine optimum number of
    cycles
   Contamination in controls, blamed
    on DNA on kimwipes.
   Contamination perfectly matched
    NS band.
Four Experiment
   Clear NS bands in control lanes!
   Controls only H2O samples.
   “What the heck is going on?!”
“There’s A Fly In My Primer!”
Fifth Experiment
   Designed to determine where in
    DNA extraction process the
    contamination was getting in
   Get leaf > Freeze leaf > Add TPS >
    Cook extract > Spin extract > Add
    DNA to PCR Tube
   Contamination was all across the
    board, including in UNOPENED PCR
    TUBES.
“There’s A Fly In My Primer!”
Other Key Observations
   Another primer pair (PHY) failed to
    work during this period of time.
   One morning the freezer door was
    slightly open; didn’t shut properly.
   Primers were diluted in H2O (no
    preservative such as EDTA)
   NS primer pair binds to rDNA
    genes, common across many
    species (not just potato).
“There’s A Fly In My Primer!”
SO... What Happened?
   One or more of the reagent tubes became
    contaminated. Likely primers.
   Contaminating organism was able to
    multiply a few cycles while the freezer
    door was open.
   Suspect contaminant was an oomycete
    fungus: has similar “NS” gene.
   Extraordinary sensitivity of PCR led to
    detection of the NS gene in the
    contaminating reagent!
“There’s A Fly In My Primer!”
Moral of the Story:
   PCR is VERY sensitive!
   Contamination with template DNA
    can come from the most unlikely
    source!
The Protocol That Never Worked
 Early PCR method development
     We were setting up our PCR lab
 Method for DNA extraction and
 PCR supplied by lab in Holland
     Hey, it’s always best to use a
      protocol that works for someone
      else, right?
 Tried method:
     Total failure. No bands at all.
The Protocol That Never Worked
 Read about PCR troubleshooting.
 Changed Conditions
     More cycles. Less cycles.
     Hotter. Cooler.
     Known good primers.
     More Mg++.
     New nucleotides.
     New polymerase.
     All failed.
The Protocol That Never Worked
 Needed “Known Good” primers and DNA to
 properly test the PCR:
     Went down the road to USDA-Albany to borrow
      some known good template DNA.
     Had primers known to work on potato DNA
      synthesized.
     IT WORKED!
 So That Was A Hint:
     Must be something to do with DNA extraction
     Either not enough DNA, or some other problem...
The Protocol That Never Worked
 Immediately requested original paper that the
 DNA extraction protocol was based on.
 Learned a LOT from reading original paper!
     Authors examined effects of all sorts of conditions:
        Freezing, temp, salt, EDTA, time, etc.
     Varying any condition by up to 50% didn’t
      substantially affect results.
        Therefore we couldn’t have screwed up the extraction.
     Comment: “Addition of EDTA requires a
      compensatory increase in the concentration of
      MgCl2.”
The Protocol That Never Worked
 Quick Calculations:
     TPS buffer is 10 mM EDTA
     We added 1.0 ul of extract, therefore 10 nmol
      EDTA.
     The protocol called for 1.0 ul of 50 mM MgCl2,
      therefore 50 nmol Mg++.
     One EDTA molecule can bind two Mg++ ions.
     Therefore, the TPS buffer was immediately
      scavenging almost half of the Mg++ we were
      adding!!!!
The Protocol That Never Worked
 Quick Experiment:
     Run a Mg++ gradient.
     No amplification with 1.0, 1.5 ul MgCl2
     Good amplification with 2.0 and 2.5 ul!
 The problem was all in the magnesium
     Actually, tried this earlier, but just happened not to
      try enough the first time...


 Never did determine why the Holland lab
 didn’t have problems.
The Protocol That Never Worked
 Moral of the Story:
     Just because a protocol works for someone else is
      NO GUARANTEE it will work for you!
        Don’t expect things to work out the first time out.
        Consider the troubleshooting to be a “learning process”;
         you’ll learn SO much MORE if things go wrong and you
         have to figure out why!
     It’s ALWAYS a good idea to go to the original
      source of the method!
        In this case, it was the original scientific paper which had
         the key to the answer.
A Typical PCR Reaction
Sterile Water         38.0   ul
10X PCR Buffer         5.0   ul
MgCl2 (50mM)           2.5   ul
dNTP’s (10mM each)     1.0   ul
PrimerFWD (25 pmol/ul) 1.0   ul
PrimerREV              1.0   ul
DNA Polymerase         0.5   ul
DNA Template           1.0   ul

Total Volume         50.0 ul

								
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