Practical molecular biology

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Practical molecular biology

• PD Dr. Alexei Gratchev
• Prof. Dr. Julia Kzhyshkowska
• Prof. Dr. W. Kaminski

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Course structure

• 10.10 Plasmids, restriction enzymes, analytics
• 11.10 Genomic DNA, RNA
• 12.10 PCR, real-time (quantitative) PCR
• 13.10 Protein analysis IHC
• 14.10 Flow cytometry (FACS)

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PCR

• Thermostable DNA polymerase
• Oligonucleotides
• dNTPs
• Buffer
• Template
• Cycling

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PCR

• Detection of pathogens
• Detection of mutations
• Person identification
• Cloning
• Mutagenesis
• and may more

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Quantification by PCR

• Ideal PCR
• Mm2N, m starting amount of template, N-number
  of cycles
• 30 cycles 230 109
• 40 cycles 1012

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Quantification by PCR

• Real PCR
• M m2N, only in the beginning of the reaction
• Critical factors
• Size of the product
• Mg concentration
• Oligonucleotide conc.
• dNTPs conc.

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End point PCR
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Real-time PCR
threshold
Ct
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Real-time PCR
threshold
Ct
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Quantification by PCR

• Measure the amount of the product after every
  cycle
• Determine threshold cycle (Ct) value for each
  sample
• Calculate the amount of the product
• Note Ct can be a fraction

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Real-time data collection

• Intercalating dyes
• Cheap
• Low specificity
• Can measure only one gene per tube
• Molecular beacons
• TaqMan probes
• Highly specific
• Several genes can be measured in one tube
  (Multiplex PCR)
• Expensive
• Multiplex PCR is hard to optimize

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Intercalating dyes

• SYBR Green

Data collected after synthesis step
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Intercalating dyes

• Denaturation analysis is needed for specificity
  analysis

One peak indicates that the reaction was specific.
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Fluorescence detection
FAM
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Fluorescence resonance energy transfer - FRET
FAM
Q
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Molecular beacons
Data collected during annealing step
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TaqMan probes
Data can be collected anytime
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Real-time PCR equipment

• Light sources
• Laser
• LED Array
• Focused halogen lamp
• Halogen lamp
• Detectors
• PMT (Photo Multiplier Tube)
• CCD camera

Light source
PMT
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Multiplexing
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Experiment planning

• Selection detection method
• Intercalating dye
• Molecular beacon
• TaqMan probe
• Selection of house keeping gene
• GAPD
• beta actin
• Selection of quantification method
• absolute (Standard curve)
• relative (ddCt)

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Absolute quantification

• The amount of template is measured according to
  the standard curve serial dilutions of known
  template (plasmid).
• Problem! Standard curve takes too much space on
  the plate.

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Relative quantification of ID3

• dCt(A) Ct(ID3 in A) - Ct(GAPD in A)
• dCt(B) Ct(ID3 in B) - Ct(GAPD in B)
• ddCt dCt( A) dCt(B)
• Relative Expression 2 -ddCt

Problem! ddCt method can be used only if both
reaction (for ID3 and GAPD) have the same
efficiency.
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Relative quantification
For ddCt the slopes of standard curves for gene
of interest and house keeping gene must be the
same.
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Relative quantification
quadruplicates
duplicates
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Relative quantification
Pipetting strategy
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Questions?