We have synthesized a number of asymmetric cyanine dyes whose basic structure is shown in Figure 9.1. Many of these dyes, generically called 'LCGreen™' dyes, can be used in PCR at saturating concentrations without inhibition. They differ in structure from the more common asymmetric cyanines (Zipper et al., 2004) by the presence of two nitrogens in the right ring, hence they are pyrimidiniums as opposed to pyridiniums. Different properties are obtained by replacing X, Rj, R2, and R3 with different moieties. Two of the most useful dyes are LCGreen™ I (introduced with the HR-1™ instrument) and LCGreen™ PLUS (introduced with the LightScanner™) both available from Idaho Technology. LCGreen™ PLUS is much brighter than LCGreen™ I and can be used on most real-time PCR machines, within the resolution constraints of each instrument. The exact
The basic structure of the LCGreen™ dye family. LCGreen™ dyes are asymmetric cyanines, different from SYBR® Green I by having two nitrogens in the right ring. Various properties are obtained by modifying the substituents X, R-,, R2, and R3. X is usually S or O. R-,, R2 and R3 are hydrogens, aliphatic or aromatic moieties that may be branched or linear, charged or uncharged. LCGreen™ I and LCGreen™ PLUS are two commercial versions of the LCGreen™ dye family.
structure of LCGreen™ I and LCGreen™ PLUS is kept a trade secret, as is the usual practice for commercial dyes in this field. The binding mode of the LCGreen™ dyes has not been investigated, but by analogy to SYBR® Green I (Zipper et al., 2004) it is likely that both intercalation and surface binding occur, with fluorescence dependent on the later.
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