Overview of the DNA Sequencing Process
The UNLV Genomics Core Facility uses the ABI Big Dye Terminator v3.1 fluorescence-based cycle sequencing kit for DNA sequencing and an ABI 3130 genetic analyzer. A pGEM 3Zf(+) control is run with every sample set to ensure instrument and technical performance. The DNA template of interest and sequencing primer are combined with the reagents of the Big Dye Terminator kit, including a modified Taq polymerase, buffer, dNTP’s, and dye-labeled ddNTP’s, and is subjected to 25 cycles of annealing, extension and denaturation in a thermal cycler, which creates and amplifies the extension products. The extension products are terminated by one of the four dye-labeled dideoxynucleotides (dye terminators) resulting in balanced populations of short and long extension products.
The unincorporated dye terminators are removed by Sephadex column purification. The extension products are then prepped and are electrokinetically injected into the capillaries. Based on their total charge the extension products are separated by size by electrophoresis. Towards the end of the capillary, the labeled fragments intercept a laser beam path and fluoresce and this fluorescence is captured by an optical detection device. Each of the four different dyes emits a unique wavelength when exposed to light which enables the sequence to be determined by ABI Data Collection v3.0 and Sequencing Analysis v.5.2 Software.