The Sequencing Reaction: Brief description

The sequencing reaction used in ABI 310 sequencing is a PCR elongation based on the Sanger dideoxy nucleotide triphosphate (ddNTP) terminator method.

In our case, we will use a reaction mix containing the four ddNTPs each conjugated with a unique fluorescent dye. As shown in Figure1 below, each dye when excited by a laser emits a specific fluorescence. This fluorescence is detected by a CCD and processed into the electropherogram where red=ddTTP, black=ddGTP, green=ddATP and blue=ddCTP.

Once we receive your sample containing DNA and the sequencing primer, we add to it the reaction mix. In addition to the dye tagged ddNTPs, this reaction mix also contains dNTPs and AmpliTAQ DNA polymerase. The sequencing elongation is carried out in a thermocycler for 25 cycles. Following the elongation reaction, sequencing products are precipitated, isolated and redissolved in sequencing running buffer. The samples are then loaded onto the ABI 310.

ABI 310 Gene Analyzer:

Sequence is obtained by fractionating the terminated extension products on a very fine 4 microliter column containing a special resin (Figure 2). The sample is drawn in electrophoretically at one end of the column and as it exits the column at the other end, it passes through a window where a laser excites the ddNTP-DYE. A CCD then detects the emission spectra used to generate the electropherogram.

At the end of the run, the column is voided as new column resin is added. The next sample is then loaded onto the column.