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Practical molecular biology PD Dr. Alexei Gratchev Prof. Dr. Julia Kzhyshkowska Prof. Dr. W. Kaminski 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) PCR Thermostable DNA polymerase Oligonucleotides dNTPs Buffer Template Cycling PCR Detection of pathogens Detection of mutations Person identification Cloning Mutagenesis and may more… Quantification by PCR Ideal PCR M=m*2N, m – starting amount of template, Nnumber of cycles 30 cycles =230 ≈109 40 cycles ≈1012 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. “End point” PCR Real-time PCR threshold Ct Real-time PCR threshold Ct 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 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 Intercalating dyes SYBR Green Data collected after synthesis step Intercalating dyes Denaturation analysis is needed for specificity analysis One peak indicates that the reaction was specific. Fluorescence detection FAM Fluorescence resonance energy transfer - FRET FAM Q Molecular beacons Data collected during annealing step TaqMan® probes Data can be collected anytime Real-time PCR equipment Light sources Laser LED Array Focused halogen lamp Halogen lamp Detectors PMT (Photo Multiplier Tube) CCD camera Light source PMT Multiplexing 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) 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. 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. Relative quantification For ddCt the slopes of standard curves for gene of interest and house keeping gene must be the same. Relative quantification duplicates quadruplicates Relative quantification Pipetting strategy Questions?
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