223- Load sample onto genetic analyzer.
- Select the capillary length, number of capillaries, and polymer
type according to the instrument user guide for creating run
modules and available materials. - Select injection time according to the instrument user guide.
Shorter amplicons can be run for shorter periods, but running
for the complete recommended time is advisable. - Start run.
- Check the quality of sequencing results before analyzing (see
Note 23). - A few free software packages are available to analyze Sanger
sequencing results. Some examples include Chromas,
Sequence Scanner, or MEGA6. - Open the chromatogram file with one of these software packages.
- A good chromatogram should appear as in Fig. 2.
- To align your sequence with the sequences available in National
Center for Biotechnology Information (NCBI) database, copy
the FASTA format from the sequence obtained and paste it in
at the “Enter Query Sequence” column at the blastn section
in Basic Local Alignment Search Tool (BLAST) (https://
blast.ncbi.nlm.nih.gov/Blast.cgi). - Click on the “align two or more sequence” option (Fig. 3 ) if
you want to compare your sequence with a template sequence
of your own rather than the NCBI database. - Fill up the rest of the form and click BLAST.
- Mutations can be spotted from a difference in the identity
between the query and subject sequence and can be confirmed
via the chromatogram (Fig. 4 ) (see Note 24).
3.6 Capillary
Electrophoresis
3.7 Sanger
Sequencing Analysis
Fig. 2 Example of good quality chromatogram. Good base calling, high intensity, and sharp peaks of the chro-
matogram are indication of a good chromatogram result
Single Gene Mutation in Esophageal Adenocarcinoma