Real-time PCR Illustrations

SYBR Green

A

A: The principle of SYBR Green detection in real-time PCR is outlined in the figure above.¹  The fluorescent dye SYBR Green is added to the PCR mixture (1).  SYBR Green is a DNA binding dye that fluoresces strongly when bound to double-stranded DNA.  At the start of the reaction, very little double stranded DNA is present, and so the fluorescent signal detected by the thermocycler is low (3).  As the reaction proceeds and PCR product accumulates, the amount of double-stranded DNA increases and with it the fluorescence signal (4-5).  The signal is only detectable during annealing and extension, since the denaturation step contains predominantly single-stranded DNA (6).

TaqMan

B

B: The principle of TaqMan real-time PCR is depicted in the schematic above.¹  The TaqMan probe is designed to be complementary to a specific sequence spanned by the PCR primers.  The TaqMan probe has a reporter dye at its 5́  end and a quencher dye at its 3́  end.  As long as the probe is intact and the reporter and the quencher dyes are in close proximity, no fluorescence signal is emitted due to the quenching effect (black arrow in 1, 2, and 3) (1).  After the annealing of the TaqMan probe (2) and the primers (3), the primers are extended by the DNA polymerase.  As the polymerase reaches the TaqMan probe, it uses its exonuclease activity to remove the probe one nucleotide at the time (4).  This releases the reporter from the proximity of the quencher and allows for the release of a fluorescence signal from the reporter (5).