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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 3
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PCR Detection of pathogens Detection of mutations
Person identification Cloning Mutagenesis and may more… 4
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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 5
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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. 6
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“End point” PCR 7
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Real-time PCR threshold Ct 8
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Real-time PCR threshold Ct 9
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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 10
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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 11
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Intercalating dyes SYBR Green Data collected after synthesis step 12
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Intercalating dyes Denaturation analysis is needed for specificity analysis One peak indicates that the reaction was specific. 13
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Fluorescence detection
FAM 14
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Fluorescence resonance energy transfer - FRET
FAM Q 15
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Molecular beacons Data collected during annealing step 16
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TaqMan® probes Data can be collected anytime 17
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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 18
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Multiplexing 19
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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) 20
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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. 21
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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. 22
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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. 23
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Relative quantification
duplicates quadruplicates 24
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Relative quantification
Pipetting strategy 25
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Questions?
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