Advanced PCR
Dave Palmer, Byotix, Inc.
Advanced PCR
PCR of Plant MaterialMultiplex PCRModifications to Standard PCRPCR Troubleshooting
Plant DNA Extraction
Rapid 3-Step Extraction Method
FreezeTPS 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 SIMULTANEOUSLYTypically 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 PCRCare in primer design Much greater chance
of primer-dimers Annealing
temperatures must be close
Much greater chance of artifacts
A Typical PCR Reaction
Component lSterile 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 lSterile Water 34.010X 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: ExampleThree primer pairs Effect, Marker, and
Internal Control genes
Control is smallest fragment, also brightest bandEffect is largest fragment, faintest band
Effect Gene
Marker Gene
Control Gene
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 difference between weak PCR reactions and nontransgenics.
Solution: Redesign primers.
Effect Gene?
Marker Gene?
Control Gene
Multiplex PCR: Example
Three primer pairs Control, marker, and
effect genes
Control gene fragment is largest and (almost) faintestEffect gene is smallest and brightest
Marker Gene
Effect Gene
Control Gene
Primers
Multiplex PCR: Example
Three primer pairs
Marker Gene
Effect Gene
Which are transgenic?
Control Gene
Primers
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
WaterBufferDNA templatePrimersNucleotidesMg++ ions DNA PolymeraseExtras
PCR Reaction Components
Water PurityContamination Amplification
Products
PCR Reaction Components
Buffer Must match polymeraseTypically contain KCl and TrisCan 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 AgeNumber of freeze-thawsContaminationAmount 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 ComponentsNucleotides 20-400 uM works well
Too much: can lead to mispriming and errors
Too much: can scavenge Mg++ Too low: faint products
AgeNumber 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 polymeraseAmount 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.Need to avoid contamination. Optimization by trial-and-error.
PCR Reaction Components
DNA Polymerase Thermostable? Activity declines with time
at 95C
Matches buffer?AgeContaminationConcentration: 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 userGlycerol, 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.htmlhttp://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html
PCR Cycling Parameters
Denaturation TempAnnealing TempExtension TempTimeNumber of CyclesReaction Volume“Odd” Protocols
PCR Cycling Parameters
Denaturation Step Must balance DNA denaturation with Taq damage95C for 30 - 60s typically is enough to denature DNATaq 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 stepCalculate 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 usefulTypically 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.htmlhttp://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 ul10X PCR Buffer 5.0 ulMgCl2 (50mM) 2.5 uldNTP’s (10mM each) 1.0 ulPrimerFWD (25 pmol/ul) 1.0 ulPrimerREV 1.0 ulDNA Polymerase 0.5 ulDNA Template 1.0 ul
Total Volume 50.0 ul