Using dyes instead of probes for genotyping has been a challenge. Early reports of using SYBR® Green I for genotyping (Marziliano et al., 2000, Pirulli et al., 2000) have been questioned (von Ahsen et al., 2001). Most subsequent work with SYBR® Green I processed the samples after PCR, including product purification and dye addition (Lipsky et al., 2001) or the addition of urea (Elenitoba-Johnson and Bohling, 2001). In our hands, it is not possible to reliably detect heteroduplexes with SYBR® Green I in a closed-tube system. When multiple duplexes are present, SYBR® Green I adequately detects high Tm products during melting, but not lower Tm products (Wittwer et al., 2003). Various reasons for this have been proposed, including dye redistribution during melting (Wittwer et al., 2003), strand reassociation during melting (Gundry et al., 2003), and G:C base pair specificity (Giglio et al., 2003). At least with small amplicons, faster melting rates (0.1-0.3°C/s) can minimize any effect of strand reassociation during melting (Gundry et al., 2003).
Dye redistribution during melting appears to be the major limitation of SYBR® Green I for heteroduplex detection. Heteroduplexes may be detected with SYBR® Green I if high enough concentrations are used (Lipsky et al., 2001). However, at these high concentrations PCR is inhibited (Wittwer et al., 1997a). The ideal dye would saturate all double-stranded DNA produced during PCR (eliminating dye redistribution) and not inhibit or otherwise adversely affect PCR.
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