practical molecular biology pd dr. alexei gratchev prof. dr. julia kzhyshkowska prof. dr. w....
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![Page 1: Practical molecular biology PD Dr. Alexei Gratchev Prof. Dr. Julia Kzhyshkowska Prof. Dr. W. Kaminski](https://reader038.vdocuments.mx/reader038/viewer/2022103004/56649cc15503460f94988c1b/html5/thumbnails/1.jpg)
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 M=m*2N, 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 ≈ m*2N, 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
PMT
Light source
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Multiplexing
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Experiment planning
Selection detection methodIntercalating dyeMolecular beaconTaqMan® probe
Selection of house keeping geneGAPDbeta actin
Selection of quantification methodabsolute (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
quadruplicatesduplicates
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Relative quantification
Pipetting strategy
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Questions?