Practical molecular biology - Methods.info protocols we trust

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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                               Light source
       LED Array                     PMT
       Focused halogen lamp
       Halogen lamp
   Detectors
       PMT (Photo Multiplier Tube)
       CCD camera
Multiplexing
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)
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




     duplicates           quadruplicates
Relative quantification
Pipetting strategy
Questions?

				
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posted:10/30/2012
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