Practical molecular biology

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

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

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

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.

7. “End point” PCR

8. Real-time PCR threshold Ct

9. Real-time PCR threshold Ct

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

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

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

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

14. Fluorescence detection FAM

15. Fluorescence resonance energy transfer - FRET FAM Q

16. Molecular beacons Data collected during annealing step

17. TaqMan® probes Data can be collected anytime

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

19. Multiplexing

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)

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.

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.

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

24. Relative quantification duplicates quadruplicates

25. Relative quantification Pipetting strategy

26. Questions?