David A. Palmer, Ph.D.

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(No Transcript)
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Real-Time PCR

• David A. Palmer, Ph.D.
• Technical Support, Bio-Rad Laboratories
• Adjunct Professor, Contra Costa College

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Objectives

• Today well talk about Real-Time PCR
• What is real-time PCR used for?
• How does real-time PCR work?
• What chemicals and instruments are used to detect
  DNA?
• What does real-time data look like?
• How can we demonstrate real-time PCR in the
  classroom?

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Part 1 What is Real-Time PCR and what is it
used for?
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What is Real-Time PCR?

• PCR, or the Polymerase Chain Reaction, is a
  process for the amplification of specific
  fragments of DNA.
• Real-Time PCR a specialized technique that allows
  a PCR reaction to be visualized in real time as
  the reaction progresses.
• As we will see, Real-Time PCR allows us to
  measure minute amounts of DNA sequences in a
  sample!

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What is Real-Time PCR?

• Conventional PCR
• tells us what.
• Real-Time PCR
• tells us how much.

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What is Real-Time PCR used for?

• Real-Time PCR has become a cornerstone of
  molecular biology. Just some of the uses
  include
• Gene expression analysis
• Cancer and Drug research
• Disease diagnosis and management
• Viral quantification
• Food testing
• Percent GMO food
• Animal and plant breeding
• Gene copy number
• Forensics
• Sample identification and quantification

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Real-Time PCR in Gene Expression Analysis

• Example BRCA1 Expression Profiling
• BRCA1 is a gene involved in tumor suppression.
• BRCA1 controls the expression of other genes.
• In order to monitor level of expression of BRCA1,
  real-time PCR is used.

Determine gene expression and publish scientific
paper!
Real-Time PCR
DNA
BRCA1
mRNA
Protein
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Real-Time PCR in Disease Management

• Example HIV Treatment
• Drug treatment for HIV infection often depends on
  monitoring the viral load.
• Real-Time PCR allows for direct measurement of
  the amount of the virus RNA in the patient.

Real-Time PCR
Viral RNA
Measure amount of virus, adjust prescriptions.
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Real-Time PCR in Food Testing

• Example Determining percentage of GMO food
  content
• Determination of percent GMO food content
  important for import / export regulations.
• Labs use Real-Time PCR to measure amount of
  transgenic versus wild-type DNA.

Real-Time PCR
wt DNA
GMO DNA
International shipments depend on results!
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Real-Time PCR in Forensics

• Example Real-Time PCR in Forensic Analysis!
• Stain Identification
• New Real-Time methods can be directly used to
  identify the composition of unknown stains, with
  much better accuracy than traditional
  color-change tests.
• DNA Quantification
• Since standard forensic STR Genotyping requires
  defined amounts of DNA, Real-Time PCR can be used
  to accurately quantify the amount of DNA in an
  unknown sample!

What is it ??
Enough DNA to ID ??
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Part 2 How does Real-Time PCR work?
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How does real-time PCR work?

• To best understand what real-time PCR is, lets
  review how regular PCR works...

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The Polymerase Chain ReactionHow does PCR work??
d.NTPs
Primers
Thermal Stable DNA Polymerase
Add to Reaction Tube
Denaturation
Annealing
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The Polymerase Chain ReactionHow does PCR work??
Extension
Extension Continued
Repeat
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The Polymerase Chain ReactionHow does PCR work??
Cycle 2 4 Copies
Cycle 3 8 Copies
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How does Real-Time PCR work?

• So thats how traditional PCR is usually
  presented.
• In order to understand real-time PCR, lets use a
  thought experiment, and save all of the
  calculations and formulas until later

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Imagining Real-Time PCR

• To understand real-time PCR, lets imagine
  ourselves in a PCR reaction tube at cycle number
  25

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Imagining Real-Time PCR

• Whats in our tube, at cycle number 25?
• A soup of nucleotides, primers, template,
  amplicons (the amplified DNA product), enzyme,
  etc.
• 1,000,000 copies of the amplicon right now.

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Imagining Real-Time PCRHow did we get here?

• What was it like last cycle, 24?
• Almost exactly the same, except there were only
  500,000 copies of the amplicon.
• And the cycle before that, 23?
• Almost the same, but only 250,000 copies of the
  amplicon.
• And what about cycle 22?
• Not a whole lot different. 125,000 copies of the
  amplicon.

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Imagining Real-Time PCRHow did we get here?

• If we were to graph the amount of DNA in our
  tube, from the start until right now, at cycle
  25, the graph would look like this

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Imagining Real-Time PCRHow did we get here?
?

• So, right now were at cycle 25 in a soup with
  1,000,000 copies of the target.
• Whats it going to be like after the next cycle,
  in cycle 26?

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Imagining Real-Time PCRSo where are we going?

• Whats it going to be like after the next cycle,
  in cycle 26?
• Probably there will be 2,000,000 amplicons.
• And cycle 27?
• Maybe 4,000,000 amplicons.

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Imagining Real-Time PCRSo where are we going?

• Whats it going to be like after the next cycle,
  in cycle 26?
• Probably there will be 2,000,000 amplicons.
• And cycle 27?
• Maybe 4,000,000 amplicons.
• And at cycle 200?
• In theory, there would be 1,000,000,000,000,000,00
  0,000,000,000,000,000,000,000,000,000,000,000,000,
  000 amplicons
• Or 1035 tons of DNA
• To put this in perspective, that would be
  equivalent to the weight of ten billion planets
  the size of Earth!!!!

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Imagining Real-Time PCRSo where are we going?

• A clump of DNA the size of ten billion planets
  wont quite fit in our PCR tube anymore!!!
• Realistically, at the chain reaction progresses,
  it gets exponentially harder to find primers, and
  nucleotides. And the polymerase is wearing out.
• So exponential growth does not go on forever!

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Imagining Real-Time PCRSo where are we going?

• If we plot the amount of DNA in our tube going
  forward from cycle 25, we see that it actually
  looks like this

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Imagining Real-Time PCRMeasuringQuantities

• How can all this be used to measure DNA
  quantities??

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Imagining Real-Time PCRMeasuringQuantities

• Lets imagine that you start with four times as
  much DNA as I do.
• Picture our two tubes at cycle 25 and work
  backwards a few cycles.

Cycle 25
Cycle Me You
25 1,000,000 4,000,000
24 500,000 2,000,000
23 250,000 1,000,000
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Imagining Real-Time PCRMeasuringQuantities

• So, if YOU started with FOUR times as much DNA
  template as I did
• Then youd reach 1,000,000 copies exactly TWO
  cycles earlier than I would!

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Imagining Real-Time PCRMeasuringQuantities

• What if YOU started with EIGHT times LESS DNA
  template than I did?

Cycle 25
Cycle Me You
25 1,000,000 125,000
26 2,000,000 250,000
27 4,000,000 500,000
28 8,000,000 1,000,000
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Imagining Real-Time PCRMeasuringQuantities

• What if YOU started with EIGHT times LESS DNA
  template than I did?
• Youd only have 125,000 copies right now at cycle
  25
• And youd reach 1,000,000 copies exactly THREE
  cycles later than I would!

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Imagining Real-Time PCRMeasuringQuantities

• We can easily see that the left-right shift in
  the curves is related to the starting quantity of
  DNA!
• Cq (Cycle Quantity) values identify the curve
  positions, based on where they cross a threshold.
• DNA Quantity and Cq value are related as
• Quantity 2Cq

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Imagining Real-Time PCRMeasuringQuantities

• We can plot the Cq value versus the Log Quantity
  on a graph

• and calculate the quantity of any unknown
  right off of the line!!

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Real-Time PCRSensitivity

• How sensitive is Real-Time PCR?
• Ultimately, even a single copy can be measured!
  In reality, typically about 100 copies is around
  the minimum amount.
• One hundred copies of a 200-bp gene is
• twenty attograms (2 x 10-17 g) of DNA!
• this is just 2/100ths of a microliter of blood!

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Part 3 How do we detect and measure DNA?
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How do We Measure DNA in a PCR Reaction?

• We use reagents that fluoresce in the presence of
  amplified DNA!

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Measuring DNA Ethidium Bromide

• Ethidium Bromide
• common and well known
• - toxic, not very bright

http//www.web.virginia.edu/Heidi/chapter12/chp12.
htm
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Measuring DNA SYBR Green I

• SYBR Green I
• Bright fluorescence!
• Low toxicity!

Ames test results from Molecular Probes Singer et
al., Mutat. Res. 1999, 439 37- 47
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Fluorescent Dyes in PCRIntercalating Dyes
SYBR Green in Action!
PCR makes more double-stranded DNA
SYBR Green dye binds to dsDNA
When illuminated with light at 490nm, the
SYBRDNA complex fluoresces at 520nm.
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Fluorescent Dyes in PCROther Options
Even more ways to detect PCR products

• Other Intercalating Dyes
• - Eva Green
• Probes
• - TaqMan Probes
• Primer/Probe Combinations
• - Scorpions
• - LUX Primers

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What Type of Instruments are used with Real-Time
PCR?

• What about the Instruments?
• Real-time PCR systems consist of THREE main
  components
• Thermal Cycler (PCR machine), linked to a
• Optical Module (to detect fluorescence in the
  tubes during the run), linked to a
• Computer (to translate the fluorescence data into
  meaningful results).

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What Type of Instruments are used with Real-Time
PCR?

• A good example is the MiniOpticon real-time
  instrument.

Optical Module Thermal Cycler Base
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What Type of Instruments are used with Real-Time
PCR?

• One more example is the Bio-Rad CFX-Touch
  real-time PCR instrument.

Optical Module Thermal Cycler Base
The CFX module scans the PCR plate with LEDs and
records fluorescence in each well at each PCR
cycle.
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What Type of Software is used with Real-Time PCR?

• The computer, running real-time software,
  converts the fluorescent signals in each well to
  meaningful data.
• Workflow
• Set up PCR protocol.
• Set up plate layout.
• Collect data.
• Analyze data.

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Part 4 What does actual real-time data look
like, and what are melt curves?
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Real-Time PCRActual Data

• This is some actual data from a recent real-time
  PCR run.
• Data like this can easily be generated by
  preparing a dilution series of DNA.

c366939
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Real-Time PCRFinal Product

• The final product of real-time PCR is a table of
  Ct values, from which amounts of DNA can be
  determined.

Well Fluor Content Cycle Quantity ( Cq )
A03 SYBR Std-1 8.90
A04 SYBR Std-2 12.20
A05 SYBR Std-3 15.34
A06 SYBR Std-4 18.77
A07 SYBR Std-5 21.84
A08 SYBR Std-6 25.24
A09 SYBR Std-7 28.82
B03 SYBR Std-1 8.85
B04 SYBR Std-2 12.12
B05 SYBR Std-3 15.31
B06 SYBR Std-4 18.69
B07 SYBR Std-5 21.76
B08 SYBR Std-6 25.24
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Real-Time PCRMelt Curves

• The fluorescence data collected during PCR tells
  us how much
• . but there is another type of analysis we
  can do that tells us what!

c366939
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Melt CurvesBasics

• Melt curves can tell us what products are in a
  reaction.
• The principle of melt curves is that as DNA melts
  (becomes single stranded), DNA-binding dyes will
  no longer bind and fluoresce.

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Melt CurvesBasics

• Melt curves can tell us what products are in a
  reaction.
• PCR products that are shorter or lower GC will
  melt at lower temperatures.
• Different PCR products will therefore have
  different shaped curves.

RFU vs Temp
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Melt CurvesTypical Data

• For convenience, we typically view the derivative
  (slope) of the actual melt curve data.
• The resulting graph looks like a chromatogram,
  with peaks that represent different PCR products.

Teaching Tip Use Melt Curves to bring up a good
discussion of why different DNA sequences will
melt at different temperatures! Talk about
base-pairing, secondary structure, energy levels,
etc!
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Melt CurvesHigh-ResolutionAnalysis

• The new field of Precision Melt Analysis even
  allows differentiation between PCR products based
  on a single-base pair mismatch!
• PMA/HRM is now used in mutation screening,
  detection of biological diversity, and genetic
  analysis.

PCR melt data from different organisms is first
collected.
Then normalized.
Then the organisms are compared against each
other.
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Part 5 How can we demonstrate real-time PCR in
the classroom?
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Crime Scene Investigator Kitin Real-Time

• CSI MATERIALS REQUIRED
• To run the CSI Kit in real-time, only a few
  additional items are needed
• iQ SYBR Green Supermix
• and appropriate tubes/caps
• A real-time PCR instrument

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Crime Scene Investigator Kitin Real-Time

• CSI INSTRUCTIONS
• An Application Note and a Starter Kit are
  available (166-2660EDU)
• The PCR reactions in the CSI kit can be run in
  real-time as an add-on to the regular kit.

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Crime Scene Investigator Kitin Real-Time

• CSI FINAL RESULTS
• The Crime Scene Real-Time Extension is great for
  first-time users!!
• Teach PCR, Real-Time, Melt Curves, etc!

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GMO Investigator Kitin Real-Time

• GMO MATERIALS REQUIRED
• To run the GMO Kit in real-time, only two
  additional items are needed
• iQ SYBR Green Supermix
• and appropriate tubes/caps
• A real-time PCR instrument

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GMO Investigator Kitin Real-Time

• GMO INSTRUCTIONS
• An Application Note and Starter Kit (166-2560EDU)
  are available
• Basically, run GMO kit with real-time reagents on
  a real-time instrument.

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GMO Investigator Kitin Real-Time

• GMO FINAL PRODUCT
• Ultimately, you can even calculate the percentage
  GMO content in actual food samples!
• The GMO Real-Time extension is perfect for more
  advanced users!

Food Plant Cq GMO Cq Delta Cq Plant Delta Cq GMO GMO Plant ddCq Ratio 2ddCt Ratio
100 GMO 23 27 0 0 0 1 100
Non-GMO 24 39 -1 -12 -11 0.0004 0.04
Product A 22 30 1 -3 -4 .0625 6.3
Product B 25 29 -2 -2 0 1 100
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Real-Time in the ClassroomGetting Started

• Getting started with teaching Real-Time PCR in
  the classroom is easy !
• Use the Crime Scene Real-Time starter kit.
• Use the GMO Investigator Real-Time kit.
• Use your own PCR primers and templates with SYBR
  Green Supermix.
• Its nothing more complicated than using a
  slightly different polymerase supermix, correct
  plastics, and running on a real-time instrument !

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Why Real-Time in the Classroom is Cool!

• Real-Time PCR in the classroom is cool for a
  number of reasons!
• Instant Results! You can see amplification
  within minutes of starting the PCR run. No
  waiting until next lab period and waiting for
  gels to run.
• What-Will-Happen-Next Factor! Because samples
  amplify at different times, many students will
  want to wait for their sample to amplify so
  they can see what happens!
• Demystifying the Black Box! Now students can see
  what happens inside the PCR machine!
• Finally a way to connect Computers and Biology!
  Many computery students get really excited to
  see the computer control of the instrument and
  data collection / analysis!

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Conclusions

• Weve covered the following topics today
• What is real-time PCR used for?
• How does real-time PCR work?
• What chemicals and instruments are used to detect
  DNA?
• What does real-time data look like?
• How can we demonstrate real-time PCR in the
  classroom?

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Resources and References

• David Palmer
• David_Palmer_at_bio-rad.com
• Bio-Rad Technical Support
• 1(800)4BIORAD
• consult.bio-rad.com
• Bio-Rad Explorer website www.explorer.bio-rad.com
  
• Bio-Rad Explorer email biotechnology_explorer_at_bio
  -rad.com
• Crime Scene Investigator PCR Basics Kit Real-Time
  PCR Application Note
• Bulletin 166-2505
• GMO Investigator Kit Real-Time PCR Application
  Note
• Bulletin 166-2605
• Real-Time PCR Applications Guide
• Bulletin 5279

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Real-Time PCRPractical Exercise!
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• Today well use the DNA in the Crime Scene Kit to
  make some dilutions for our real-time experiment!
• Each workgroup will have DNA from the Crime Scene
  kit that has been diluted 110, 1100, 11000,
  110000, or undiluted. This is your Unknown
  DNA.
• Each workgroup will prepare four real-time PCR
  reactions
• Unknown DNA (replicate 1)
• Unknown DNA (replicate 2)
• Unknown DNA diluted 1100 (replicate 1)
• Unknown DNA diluted 1100 (replicate 2)
• If all goes well, youll be able to tell from the
  Cq values
• Which unknown DNA you started with,
• How accurate your pipetting is,
• Whether your mini-dilution series demonstrates
  high-efficiency PCR.

Todays Experiment An Overview
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• Step 1 DNA Dilutions
• Dilute your Unknown DNA 1100.
• Mix 1 ul of your DNA (screw-cap tube labelled
  1-5) into 99 ul of water (screw-cap tube labelled
  W).
• Step 2 Prepare your PCR Tubes
• Add 20 ul of the spiked SYBR Green Supermix (from
  the screw-cap) tube labelled SMX to four PCR
  tubes.
• Step 3 Add DNA to your PCR Tubes
• Add 20 ul of your DNA samples to each PCR tube
• Unknown Replicate A
• Unknown Replicate B
• Unknown 1100 Replicate A
• Unknown 1100 Replicate B
• Mix gently, avoiding bubbles!
• Label appropriately.
• Step 4 Cap and Load the PCR Tubes
• Place the optically-clear flat caps on the tubes.
• Place your reactions in the real-time PCR
  machine.

Todays Experiment Step-By-Step
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• Our PCR protocol will look like this
• 1. 95C for 3 min (activates Taq)
• 2. 95C for 10 sec (denatures)
• 3. 52C for 30 sec (extend / anneal)
• 4. Plate read (captures fluorescence data)
• 5. Goto Step 2 for 39 more times

Todays Experiment PCR Protocol
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Real-Time PCR

• David A. Palmer, Ph.D.
• Technical Support, Bio-Rad Laboratories
• Adjunct Professor, Contra Costa College

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BONUS How do we optimize Real-Time PCR and
troubleshoot problems?
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• Optimization of real-time PCR reactions is
  important
• Since real-time PCR calculations are based on a
  doubling of product every cycle, if the reaction
  isnt optimized, this doubling will not occur.

Optimization Why?
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• A well-optimized reaction will have evenly spaced
  standard curves with tight replicates
• At 100 efficiency, 10-fold serial dilutions will
  be spaced 3.3 cycles apart from each other.

Optimization Example
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• Optimization is normally done as follows
• Design multiple primer sets.
• Empirically test each primer set with a standard
  curve.
• Select best primer set, then run a temperature
  gradient experiment to determine best annealing
  temperature.
• Standard curves are ideal for assessing
  optimization.

Optimization Basics
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• Why is it important for teachers to be able to
  solve real-time PCR problems?
• Help students have better success with their
  projects!
• Preventing problems at the start can help avoid
  lost experiments and reagents!
• Being able to explain unusual results leads to
  great teaching opportunities!

Trouble-shooting Skills Why?
Teaching Tip Very often a students first
real-time data isnt perfect this makes for a
great chance to teach better pipetting skillls,
experimental design, etc!
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Trouble-Shooting

• A successful real-time PCR experiment will have
  the following characteristics

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Trouble-ShootingReplicates

• If replicates arent tightly clustered, suspect
• Pipetting error
• Poorly optimized PCR reactions
• Sample evaporation
• Unknowns outside of range of detection
• Instrument calibration

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Trouble-ShootingBaselines

• If baselines arent flat, suspect
• Sample evaporation
• Bubbles
• Reagents not thoroughly mixed
• Baseline window not properly set

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Trouble-ShootingDilutions

• If the dilution series comes out compressed or
  stretched, suspect
• Pipetting
• Too much DNA (for your assay)
• PCR inhibitors
• Too little DNA (for your assay)
• Poor PCR efficiency

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Trouble-ShootingCurve Shape

• If curves are not S-shaped, suspect
• Curves are not actual PCR products!
• Sample evaporation
• Fluorescence drift in unamplified samples
• Something seriously wrong with assay

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Trouble-ShootingCurve Shape

• If curves are not smooth, suspect
• Poor pipetting (bubbles)
• Sample evaporation
• Poor assay (low fluorescence reagents)
• System malfunction (line noise)

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Trouble-ShootingMelt Peaks

• If melt curves have more than one peak
• More than one product
• Possible normal primer-dimers
• Using too low an annealing temperature
• Primers need to be redesigned

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Trouble-Shooting

• Common themes in troubleshooting
• Care in pipetting.
• Care in choice of plastics and sealing the
  plates.
• Care in experimental design.
• Use of Positive and Negative Controls.

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Teaching Tip Use Real-Time PCR to teach the
importance of properly designed experiments !!
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Real-Time PCR

• David A. Palmer, Ph.D.
• Technical Support, Bio-Rad Laboratories
• Adjunct Professor, Contra Costa College