Q-PCR

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Q-PCR

• Bige Vardar -01780333

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OUTLINE

• What is PCR and purpose of it?
• What is Q-PCR and purpose of it?
• How does Q-PCR work?
• Types of Q-PCR probes and comparison of types
• Advantages and Disadvantages of Q-PCR vs. PCR
• Questions

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What is PCR?

• Stands for Polymerase Chain Reaction
• copies of DNA fragment(X)Xo(1E)n where nof
  cycles in PCR reaction and Eefficiency
• Steps in PCR
• Denaturation(95oC)1min
• Annealing(55oC)45sec
• Extension(72oC)2min
• Cycle thorugh 25-35

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Purpose of PCR

• Easy to sequence some of million copies and
  detect rather than trying to sequence and detect
  a single copy of a gene
• Can calculate which sample is biggest when
  comparing two or more DNA fragments
• It is used to clone specific genes

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What is Q-PCR?

• Stands for Quantitative Polymerase Chain Reaction
• Assay that monitors accumulation of DNA from a
  PCR reaction
• Important technique to quantify RNA(mRNA) levels
  and DNA gene levels in biological samples
• Templates DNA, cDNA,RNA
• Similar to PCR except the progress is monitored
  by a camera or detector
• Uses fluorescence-based probes to detect DNA or
  RNA
• Data collection start at early exponential phase
  and examined at the same time with detection

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Research Objectives

• Gene validation
• Primary validation
• Confirmation of microarray data
• Viral detection
• Bacterial detection and identification
• Gene duplication or DNA quantification
• www.scienceboard.net

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Applications

• Pathogen detection
• GMO analysis
• Quality control
• Forensics
• Methylation studies
• Detect proteins with Q-PCR
• DNA/RNA quantification
• Protein stability testing
• Drug therapy efficacy / drug monitoring

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Q-PCR

• Assay uses a standard curve to quantitate the
  amount of target present using a
  fluorescence-labeled probe for detection.
• Each technique uses some kind of fluorescent
  marker which binds to the DNA

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Types of Q-PCR

• Hydrolyzation based Assays
• Taqman, Beacons, Scorpions
• DNA-binding agents
• SYBR Green
• Hybridization based Assay
• Light cycler(Roche)

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Taqman Probes

• Fluorescence-labeled oligonucleotides (TaqMan
  probes)
• TaqMan probes are complementary to a region of
  the target gene
• The 5' to 3' exonuclease activity of the
  polymerase cleaves the probe, releasing the
  fluorophore into solution

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Characteristics of Taqman Probes

• Oligonucleotides longer than the primers (20-30
  bases long with a Tm value of 10 oC higher) that
  contain a fluorescent dye usually on the 5' base,
  and a quenching dye typically on the 3' base
• The excited fluorescent dye transfers energy to
  the nearby quenching dye molecule rather than
  fluorescing(FRET)
• Uses universal thermal cycling parameters and PCR
  reaction conditions
• One specific requirement for fluorogenic probes
  is that there be no G at the 5' end

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Molecular Beacons

• Contain fluorescent and quenching dyes at either
  end but they are designed to adopt a hairpin
  structure while free in solution to bring the
  fluorescent dye and the quencher in close
  proximity for FRET to occur
• Have two arms with complementary sequences that
  form a very stable hybrid or stem

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molecular beacons
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SYBR Green I

• A fluorogenic minor groove binding dye that
  exhibits little fluorescence when in solution but
  emits a strong fluorescent signal upon binding to
  double-stranded DNA
• Binds to the minor groove of the DNA double helix
  with a higher affinity for dsDNA than for
  single-stranded DNA (ssDNA)
• Fluorescence is greatly enhanced (1000-fold) upon
  DNA-binding making this dye a sensitive indicator
  for the quantity of dsDNA

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• http//www.youtube.com/watch?v5ZEySHfCWAUfeature
  related

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Taqman vs. SYBR Green I

• SYBR Green
• Advantages
• Relative low cost of primers.
• No fluorescent-labeled probes required.
• Disadvantages
• Less specific only primers determine
  specificity.
• Specific and non-specific double-stranded PCR
  products generate the same fluorescence signal
  upon binding SYBR Green I dye.
• Not possible to multiplex multiple gene targets.

• TaqMan Probe
• Advantages
• Increased specificity
• Use when the most accurate quantitation of PCR
  product accumulation is desired.
• Option of detecting multiple genes in the same
  well (multiplexing).
• Disadvantages
• Relative high cost of labeled probe.

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Q-PCR vs. PCR

• Some of the problems with End-Point Detection
• Poor Precision
• Low sensitivity
• Short dynamic range lt 2 logs
• Low resolution
• Non - Automated
• Size-based discrimination only
• Results are not expressed as numbers
• Ethidium bromide for staining is not very
  quantitative
• Post PCR processing

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Eventually the reactions begin to slow down and
stop all together or plateau.Each tube or
reaction will plateau at a different point, due
to the different reaction kinetics for each
sample. These differences can be seen in the
plateau phase.The plateau phase is where
traditional PCR takes its measurement, also known
as end-point detection.
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• Hard to differentiate between the 5-fold change
  on the Agarose gel. Q-PCR is able detect a
  two-fold change (i.e. 10 Vs. 20 copies).

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BioRad iCycler
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References

• Dorak MT (Ed) Real-Time PCR (Advanced Methods
  Series). Oxford Taylor Francis, 2006
  http//dorakmt.tripod.com/genetics/realtime.html
• http//www.protocolonline.org/prot/Molecular_Biolo
  gy/PCR/Real-time_PCR/index.html
• SYBR Green Quantitative PCR Protocol
  http//www.genetics.ucla.edu/labs/lusis/greenquant
  itative.htm
• Quantification using real-time PCR
  technologylthttp//www.wzw.tum.de/gene-quantificati
  on/klein-2002.pdfgt
• Real-Time PCR (qPCR) Basics lthttp//www.primerdes
  ign.co.uk/Download20material/Beginners20guide20
  to20real-time20PCR.pdfgt

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QUESTIONS?