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The BigDye terminators are labelled with the following dRhodamine acceptor dyes:
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BigDye is a set of dye terminators labelled with novel, high-sensitivity dyes [175]. The new dye structures contain a fluorescein donor dye, e.g., 6-carboxyfluorescein (6-FAM), linked to one of four dichlororhodamine acceptor dyes. The excitation maximum of each dye label is that of the fluorescein donor, and the emission spectrum is that of the dRhodamine acceptor (see Figure 4.3).
The donor dye is optimised to absorb the excitation energy of the argon ion laser. The linker affords extremely efficient energy transfer (quantum efficiency nearly 1.0) between the donor and acceptor dyes.
The BigDye terminators also have narrower emission spectra than the rhodamine dye terminators, giving less spectral overlap and therefore less noise. The brighter signal and decreased noise provides an overall 4–5Õ gain in signal-to-noise ratio. The dye terminator structures are shown in Figure 4.2.
The used BigDye terminator Cycle Sequencing Kit includes dye terminators, deoxynucleoside triphosphates, AmpliTaq DNA Polymerase, FS, rTth pyrophosphatase, magnesium chloride, and buffer. The dNTP mix includes dITP in place of dGTP to minimise band compressions. The dNTP mix also uses dUTP in place of dTTP. dUTP improves the incorporation of the T terminator and results in a better T pattern.
BigDye terminator cycle sequencing was done in a GeneAmp PCR System 9700 (Perkin-Elmer) using simulated tube control. 0.5Õ reactions (10 µl) were prepared using 3 pmol sequencing primer (Table 4.8), containing approximately 3 – 10 ng of PCR template. Samples were sequenced in both directions.
Amplification was over 25 cycles at 96℃C for 10 seconds, 50℃C for 5 seconds, and 60℃C for 4 minutes.
The amplification products were ethanol precipitated for 10-15 minutes at room temperature, centrifuged at maximum speed for 20 minutes, washed with 250 µl 70% ethanol and dried by putting the open tubes back into the thermal block at 90℃C for one minute.
A different protocol was used for amplification in 96-well trays. 8 µl of deionised water and 32 µl of 95% ethanol were pipetted in each tube. The tray was sealed with Scotch tape, inverted a few times to mix properly, and left at room temperature for 15 minutes to precipitate the extension products. The tray was placed in a table-top centrifuge equipped with tube-tray adaptors and spun for 45 minutes at 2000g. Supernatant was discarded by inverting the opened tray onto a paper towel. The inverted tray with the paper towel was placed into the centrifuge and spun at 700g for 1 minute. After removing the tray from the centrifuge and discarding the paper towel, samples were ready for loading (see section 4.2.8).
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© 2001 Alexander Binder
