Israel maritime college qPCR Molecular lab

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.

qPCR Techniques SYBR® Green technique The TaqMan® probe technique

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

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

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

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)

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!

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

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