1. Practical molecular biology
PD Dr. Alexei Gratchev
Prof. Dr. Julia Kzhyshkowska
Prof. Dr. W. Kaminski

2. 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)

3. PCR Thermostable DNA polymerase Oligonucleotides dNTPs Buffer Template
Cycling 3

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

5. 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

6. 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

7. “End point” PCR 7

8. Real-time PCR
threshold Ct 8

9. Real-time PCR
threshold Ct 9

10. 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

11. 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

12. Intercalating dyes
SYBR Green
Data collected after synthesis step 12

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

14. Fluorescence detection
FAM 14

15. Fluorescence resonance energy transfer - FRET
FAM Q 15

16. Molecular beacons
Data collected during annealing step 16

17. TaqMan® probes
Data can be collected anytime 17

18. 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

19. Multiplexing 19

20. 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

21. 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

22. 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

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

24. Relative quantification
duplicates
quadruplicates 24

25. Relative quantification
Pipetting strategy 25

26. Questions?