Automated Sequencing of the psaB Gene Using LI-COR 4200 DNA Analyzer
Written by Erin Downey
A section of cloned DNA can be sequenced by a variety of methods, the most common of which is the Sanger method. The Sanger method uses low concentrations of dideoxynucleoside triphosphates (ddNTPs) to act as chain terminators. These are used with the normal, unmodified deoxynucleoside triphosphates. Terminators are separated into ddATP, ddTTPs, ddCTPs, and ddGTPs samples so that when they are sequenced, each lane identifies the terminal nucleotide. This results in fragments of varying sizes that can be resolved on a sequencing gel that allows the DNA to be read by the LI-COR 4200 DNA Analyzer. Analysis is accomplished through the addition of fluorescent dye to the DNA samples, which are loaded into a polyacrylamide gel.
MATERIALS AND METHODS
The protocol outlined in “ AUTOMATED SEQUENCING OF THE psaB GENE” provided by Dr. Mary U Connell, Biology Department, Appalachian State University, was followed. Ambiguities were edited using Align IR.
Sequences for both the 700 and 800 channel were obtained, the 700 sequence is shown in Figure 1 and the 800 sequence is shown in Figure 2. The 700 channel sequence had a total read length of 807 bases with only 16 ambiguities. The 800 channel sequence had a read length of 798 with only 32 ambiguities. The ambiguities were edited in Align IR by comparing multiple runs. The results were analyzed by searching in the Basic Local Alignment Search Tool (BLAST) database provided the National Institute of Health (NIH). The most analogous sequence belonged to Pylaiella littoralis. The Contig sequence is show in Figure 3.
The psaB gene was isolated from Scytosiphon lomentaria, a cousin to the P. littoralis identified in the BLAST search. This indicative of a successful sequencing of the gene since there was high homology between the experimental and the database. The Contig diagram in Figure 3 was important to show that there was overlap between the sequenced areas. Without overlap, it would be impossible to create a contiguous sequence of the entire gene.