Working With SYBR Green

Considerations when using SYBR Green
The most common detection methods when performing Real-Time PCR are SYBR and TaqMan.

TaqMan is Fluorogenic-labeled probes that use the 5′ nuclease activity of Taq DNA polymerase. The master mix which bases its detection on 5’ nuclease activity requires three hybridization events: two PCR primers and one probe, with hydrolysis of the probe during extension of the primers.

SYBR® Green is a non-specific molecule that binds to double-stranded DNA resulting in a significant increase in its fluorescent intensity. This method is based on two hybridization events, the forward and reverse primers.

Regardless of the binding method, there are two requirements for a DNA binding dye for real-time detection of PCR:

  • Increased fluorescence when bound to double-stranded DNA
  • No inhibition of PCR

SYBR dye detects polymerase chain reaction (PCR) products by binding to double-stranded DNA formed during PCR.

When SYBR dye is added to a sample, it immediately binds to all double-stranded DNA present in the sample. During PCR, DNA polymerase amplifies the target sequence which creates the PCR products. SYBR dye then binds to each new copy of double-stranded DNA. As the PCR progresses, more PCR product is created. SYBR® dye binds to all double-stranded DNA, so the result is an increase in fluorescence intensity proportioned to the amount of PCR product produced.

Advantages of SYBR dye

  • It can be used to monitor the amplification of any double-stranded DNA sequence.
  • No probe is required, which can reduce assay setup and running costs, assuming that your PCR primers are well designed, and your reaction is well characterized.

Disadvantages of SYBR dye

The primary disadvantage is that it may generate false positive signals, i.e., because the SYBR dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences. Therefore, it is extremely important to have well-designed primers that do not amplify non-target sequences, and that melt curve analysis be performed.

Additional consideration

Another aspect of using DNA binding dyes is that multiple dye molecules may bind to a single amplified DNA molecule.

A consequence of multiple dye binding is that the amount of signal is dependent on the mass of double-stranded DNA produced in the reaction. Thus, if the amplification efficiencies are the same, amplification of a longer product will generate more signal than a shorter one.

This contrasts with the use of a fluorogenic probe in Taqman method, in which a single fluorophore is released from quenching for each amplified molecule synthesized, regardless of its length.

SYBR mixes family has new, unique and exciting member!

Please meet the PowerTrack™ SYBR™ Green Master Mix, which changes its color in different stages of your plate preparation!

The new PowerTrack™ SYBR helps reduce pipetting errors and allows reproducibility, enable ease of use and provide flexibility at a competitive price.

Features of PowerTrack SYBR Green Master Mixes include:

  • Two-color tracking dye system indicates where pipetting has occurred
  • Broad primer Tm and primer concentration compatibility allows flexibility in qPCR reaction setup with minimal optimization
  • Superior specificity and tight reproducibility in Ct values over a broad dynamic range improve data quality
  • Compatible with SuperScript IV VILO master mix reverse transcription for fast, reproducible results
  • Formulated with UNG/dUTP to prevent contamination of carryover PCR products
  • Broad instrument compatibility