SYBR Green Real Time PCR

Real Time PCR Detection Fluorescent Primer Dyes - SYBR Green.

Real Time PCR Fluorescent Primer Dyes - SYBR Green

In real time PCR, a fluorescent dye called SYBR Green is often used to monitor DNA synthesis.

SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to double-stranded DNA. The resulting DNA-dye-complex absorbs blue light ((lambda max = 498 nm) and emits green light (lambda max = 522 nm). SYBR Green I could replace the very strong mutagen ethidium bromide.

Diagram 1: A Spectrogram of SYBR Green I showing the fluorescence excitation and emission spectra of SYBR Green I bound to DNA.

SYBR Green binds to double stranded DNA but not to single-stranded DNA and is frequently used to monitor the synthesis of DNA during real-time PCR reactions.

When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide does, which is why we use Sybr Green rather than ethidium bromide; we also use Sybr green because the ratio of fluorescence in the presence of double-stranded DNA to the fluorescence in the presence of single-stranded DNA is much higher that the ratio for ethidium bromide).

Other methods can also be used to detect the product during real-time PCR, but will not be discussed here. However, many of the principles discussed below apply to any real-time PCR reaction.

Figure 1: The chemical structure of SYBR Green I.

SYBR Green Real Time PCR References

Zipper, H. et al.  (2004).  Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications.  Nucleic Acids Res.  32, e103.

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