1. Israel maritime college
qPCR
Molecular lab

2. qPCR
A technique based on PCR reaction for the detection and quantification of target DNA molecule.
Allows the scientist to actually view the increase in the amount of DNA as it is being amplified.
There are several real-time PCR methods, all are based on fluorescent signal analysis being released during the run.

3. qPCR Techniques
SYBR® Green technique
The TaqMan® probe technique

4. SYBR® Green technique It binds to any dsDNA
SYBR Green is a double stranded nucleic acid
binding dye.
It binds to any dsDNA
Can be used as a tool for microorganisms observation

5. SYBR® Green technique:
The SYBR green floats unbounded with no significant fluorescence signal
Dye molecules can bind only to a double strand. DNA binding results in a dramatic increase of fluorescence signal. The resulting DNA-dye-complex absorbs blue light (λmax = 488 nm) and emits green light (λmax = 522 nm).
During elongation, more and more dye molecules bind to the newly synthesized DNA

6. Melt curve analysis The specific products Non-specific products
Primer dimer <75°C

7. The specific product, non-specific products and primer dimers are detected as equally well
number of ways to handle this problem
Hot start techniques like TaqStart antibody can be helpful in reducing primer dimer
Melting curve analysis of the reaction.  This can help to determine the fraction of the signal coming from the desired product and the fraction coming from primer dimer
Careful optimization of PCR conditions can usually reduce primer dimers (annealing temp., primer concentration)

8. The TaqMan® technique
The probe is assembled from a fluorescence reporter and a quencher.
Important:
The annealing temp. of the probe have to be higher than the annealing temp. of the primers!

9. Quantitative PCR Primers used: malB gene, DNA of E. coli k12 MG1655
Threshold line
Primers used: malB gene, DNA of E. coli k12 MG1655

10. Gene expression
Primers for prmB, wcaH and thrS genes, DNA of E. coli strains from sewage