DNA Quantitation and RealTime PCR

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DNA Quantitation and Real-Time PCR

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Quantitation Overview
quantitate tr.v. to determine or measure the
quantity of.

• Why quantitate?
• Methods for quantitation
• Spectrophotometry
• Slot Blot (Quantiblot)
• Microtiter Plate
• Real Time PCR

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DNA Quantitation

• Following DNA extraction, an unknown
  concentration of DNA and other substances exist
  in the extract.

How much DNA is in the sample?
Why do we care?
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DNA Quantitation

• Quantitation can indicate whether an extraction
  was successful, but

Amplification of STRs or mtDNA can be
accomplished on samples containing DNA at a level
below the detection limit of most quantitative
techniques.
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DNA Quantitation

• Main reason for quantitation

Most PCR assays work best when DNA is at a
certain level.

• Too little DNA extract
• Failure to amplify any DNA
• Allele dropout (incomplete amplification)

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DNA Quantitation

• Main reason for quantitation

Most PCR assays work best when DNA is at a
certain level.

• Too much DNA extract
• Amplified STR peaks off scale
• Increase in PCR inhibitors

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DNA Quantitation

• Spectrophotometry

• DNA absorbs UV light at 260 nm
• More DNA More absorbance
• Not specific to human DNA

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DNA Quantitation

• Yield Gel

• DNA separated on agarose gel
• Stained with ethidium bromide (requires double
  stranded DNA)
• Not specific to human DNA

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DNA Quantitation

• Microtiter plate

• Picogreen is interchelating dye.
• Binds between base pairs in double stranded DNA,
  enhancing fluorescence.
• Fluorescence related to amount of DNA in solution.

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DNA Quantitation

• Microtiter plate

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DNA Quantitation

• Slot Blot Analysis

• DNA captured on membrane.
• Labeled probe bound to DNA
• Intensity of label related to quantity of DNA.

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DNA Quantitation

• Slot Blot Analysis

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DNA Quantitation

• Capturing DNA on membrane

• DNA mixed with spotting solution to give the
  sample color
• Biodyne Membrane prewet. Ready to bind DNA.
• DNA/spotting solution applied to membrane using
  slot blot apparatus.

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DNA Quantitation

• Binding Probe (D17Z1)

• 5-biotin-TAGAAGCATTCTCAGAAACTACTTTGTGATGATTGCATTC
  -3
• Probe only binds to human DNA (chromosome 17)
• Biotin has high affinity for many proteins,
  including Streptavidin.

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DNA Quantitation

• Adding HRP-SA

• Horseradish peroxidase-streptavidin complex added
• Streptavidin binds biotin (probe)
• Horseradish peroxidase will play part in color
  changing reaction

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DNA Quantitation

• Adding TMB H2O2

• TMB will change color where HRP is
• Color intensity relates to the amount of HRP,
  which relates to the amount of DNA

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Real Time PCR

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

18
Summary

• Chemistry
• What are the reaction components?

Instrumentation What amps and detects?
Quantitation Applications Forensics
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Chemistries

• Generic
• Dyes bind to dsDNA
• Includes intercalating dyes
• Non-specific

• Strand-specific (Quantifiler)
• Binds only to sequence being amplified

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Real Time PCR

• SYBR Green Detection

• Type of generic reaction SYBR green
  non-specifically binds all dsDNA

• Increased fluorescence when bound to DNA

• More double stranded DNA more binding more
  fluorescence

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Real Time PCR

• SYBR Green Detection

• Fluorescence measures amount of amplicon during
  PCR

• Forensically, can be used to calculate how much
  DNA was present before reaction.

• Quant method very similar to strand specific
  binding (later)

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Real Time PCR

• Strand-Specific Detection

• Fluorescent labeled probe (sequence specific)
  binds to DNA
• More PCR product more sequence for probe to
  bind with
• Only fluoresces during/after it is bound
  (different mechanisms)

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Real Time PCR

• Strand-Specific Detection (Mechanisms)

Unimportant Dual hybridisation, molecular
beacon, hybeacons, etc. Important 5 Nuclease
Activity
5 Nuclease Activity based probes will fluoresce
after binding when the 5 end is cleaved by Taq
polymerase.
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Real Time PCR

• 5 Nuclease Activity

Reporter (FAM)
Quencher (TAMRA)
R
Q
Probe
Why doesnt the probe fluoresce when unbound?
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No blue fluorescence when unbound
Dye FAM
In case of TAMRA, decays by fluorescing at a
different ?
Q TAMRA
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Real Time PCR

• 5 Nuclease Activity

Reporter (FAM)
Energy transfer
Quencher (TAMRA)
R
Q
Probe
488nm
Why doesnt the probe fluoresce when unbound?
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Real Time PCR

• 5 Nuclease Activity

Reporter (FAM)
Quencher (TAMRA)
R
Q
Probe
Why does the probe fluoresce when bound?
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Blue fluorescence when bound/cleaved
Real Time PCR
Dye physically separated from Quencher
Dye FAM
Q TAMRA
Allowed to fluoresce
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Taqman

• Taqman describes technology of probe using
  Reporter/Quencher

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Taqman

• Probe binds DNA during cool down before primers.

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Taqman

• Reporter is cleaved from probe. Fluoresces

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Taqman

• Reporter is cleaved from probe. Fluoresces

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Real Time PCR

• 5 Nuclease Activity

Reporter (FAM)
Blue light detected
R
Quencher (TAMRA)
Q
488nm
Probe
Why does the probe fluoresce when bound?
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Real Time PCR

• Strand-Specific Detection (Probes)

• Probe should bind before primers
• Tm should be 5ºC higher than primers
• Probe usually 20-30 bp long

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Instrumentation

• Real Time thermal cyclers have capability to
  excite fluorophore and detect emitted light.

• Many manufacturers make real-time PCR thermal
  cyclers.

• Forensics currently dominated by Applied
  Biosystems 7000 and 7900HT.

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ABI 7900HT is high throughput.
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Instrumentation

• 7900HT

Laser Argon ion, 488nm Detector CCD camera
(500 - 660nm)
Uses 96 and 384 plates
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Real Time PCR

• Quantifiler and Quantifiler Y

Designed for Quantitation of human DNA or human
male DNA
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Quantifiler Human
Primer 1
R
Q
dNTPs
Probe
Primer 2
Tris Buffer
MgCl2
Known amount DNA
Taq Polymerase
PCR Components
Internal PCR Control (IPC)
R
Q
Primer 1
Probe
Primer 2
Primers Human Telomerase Transcriptase gene
Taqman Probe Human Telomerase Transcriptase gene
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Quantifiler Y Human Male
Primer 1
R
Q
dNTPs
Probe
Primer 2
Tris Buffer
MgCl2
Known amount DNA
Taq Polymerase
PCR Components
Internal PCR Control (IPC)
R
Q
Primer 1
Probe
Primer 2
Primers SRY gene
Taqman Probe SRY gene gene
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Real Time PCR gt Quantifiler

• Positive Result

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Real Time PCR gt Quantifiler

• Negative Result

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Real Time PCR gt Quantifiler

• PCR Inhibition

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Real Time PCR gt Quantifiler

• X 100,000 copies
• ? 1000 copies

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Threshold too high

• Standard curve uses the cycle number where
  fluorescence crosses threshold.
• Threshold should be set as low as possible
  accuracy higher for early measurements

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Threshold good

• Standard curve uses the cycle number where
  fluorescence crosses threshold.
• Threshold should be set as low as possible
  accuracy higher for early measurements

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• X 100,000 copies
• ? 1000 copies

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• If it crossed at 15

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• If it crossed at 15 about 7E05 copies of DNA in
  extract.

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Quantitation

• Can also be reported in amount of DNA (ng/uL like
  Quantiblot)
• Since it is detecting viable sequence, amount of
  DNA will decrease with degradation

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Web Animation

• http//www.vetscite.org/issue1/tools/leute_2_0800.
  htm