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 3

PCR Detection of pathogens Detection of mutations Person identification Cloning Mutagenesis and may more… 4

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

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

“End point” PCR 7

Real-time PCR threshold Ct 8

Real-time PCR threshold Ct 9

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

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

Intercalating dyes SYBR Green Data collected after synthesis step 12

Intercalating dyes Denaturation analysis is needed for specificity analysis One peak indicates that the reaction was specific. 13

Fluorescence detection FAM 14

Fluorescence resonance energy transfer - FRET FAM Q 15

Molecular beacons Data collected during annealing step 16

TaqMan® probes Data can be collected anytime 17

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

Multiplexing 19

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

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

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

Relative quantification For ddCt the slopes of standard curves for gene of interest and house keeping gene must be the same. 23

Relative quantification duplicates quadruplicates 24

Relative quantification Pipetting strategy 25

Questions?