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