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?