practical molecular biology pd dr. alexei gratchev prof. dr. julia kzhyshkowska prof. dr. w....
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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, N-
number 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
PMT
Light source
Multiplexing
Experiment planning
Selection detection methodIntercalating dyeMolecular beaconTaqMan® probe
Selection of house keeping geneGAPDbeta actin
Selection of quantification methodabsolute (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
quadruplicatesduplicates
Relative quantification
Pipetting strategy
Questions?
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