Dye Primer Cycle Sequencing

4.2.5 Dye Primer Cycle Sequencing

The fluorescein/rhodamine dye primers used have the following dye labels:


Primer	Dye Label

A	JOE
C	5-FAM
G	TAMRA
T	ROX

Table 4.5:

Fluorescein/rhodamine dye labels

The structures of the fluorescein/rhodamine dye primers are shown in Figure 4.1. Note that 5-FAM and JOE are fluorescein dyes and TAMRA and ROX are rhodamine dyes.

Figure 4.1:

Fluorescein/rhodamine dye primers

The used ABI PRISM Dye Primer Cycle Sequencing Kit includes AmpliTaq DNA Polymerase FS, dye-labelled M13 forward and reverse primers and all the required components for the sequencing reaction. The deoxy- and dideoxynucleotide ratios in the dNTP mix have been optimised to give a balanced distribution of signal between base 10 and base 700+ after the primer. The dNTP mix includes 7-deaza-dGTP in place of dGTP to minimise band compressions.

Dye Primer Sequencing Protocol

Dye Primer cycle sequencing was done in an OmniGene Thermocycler (Hybaid) using tube control. 0.5Õ reactions were prepared for all four dye labelled reactions containing approximately 5 – 10 ng of PCR sample and overlaid with 10 µl of light mineral oil. The thermal protocol used is given in Table 4.6.


Step	Action

1	Repeated for 15 cycles:
	96℃C for 10 sec.
	55℃C for 5 sec.
	70℃C for 1 min.

2	Repeated for 15 cycles:
	96℃C for 10 sec.
	70℃C for 1 min.

Table 4.6:

Dye Primer cycle sequencing protocol

The amplification products were transferred into one tube, ethanol precipitated for 10-15 minutes, centrifuged at maximum speed for 20 minutes and dried by putting the open tubes back into the thermal block at 90℃C for one minute.

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