Quick Start Guide

DNA Sequencing of COI amplicons

Background DNA sequencing is a procedure for determining the order in which nucleotides (adenine, guanine, cytosine, and thymine) appear in an individual gene or gene fragment, a continuous cluster of genes, a complete chromosome, or an entire genome. Over the last several decades, a variety of sequencing methods have been developed for different applications and research goals. A researcher’s selection of a particular method is based on a variety of considerations, including speed, cost, accuracy, and the length of the DNA molecule to be sequenced. Dye terminator cycle sequencing – an automated variation of Sanger sequencing – is the method of choice for DNA barcoding. This PCR-based method of automated DNA sequencing is performed at a nominal cost by both commercial and university-based sequencing facilities.

Methodology For DNA barcoding, two dye terminator sequencing reactions are performed separately for each COI amplicon. The forward sequencing reaction will determine the nucleotide sequence of the sense strand, whereas the reverse sequencing reaction will determine the nucleotide sequence of the antisense strand.
The following components are common to both the forward and reverse sequencing reactions, which are performed in separate tubes: 1) multiple copies of a double- stranded COI amplicon (the DNA template for each sequencing reaction); 2) a heat- stable DNA polymerase; 3) dNTPs (the basic building blocks of DNA); and 4) ddNTPs (fluorescently labeled terminator nucleotides that lack an –OH group at position 3 of the ribose ring). Unlike conventional PCR, only a single oligonucleotide primer is used for each sequencing reaction.
Each sequencing reaction progresses through the same major steps of a PCR reaction:

1. During the denaturation step, each sequencing reaction mixture is heated to ~96˚C to disrupt the hydrogen bonds that hold the sense and antisense strands of the COI amplicon together.
2. During the annealing step, each reaction mixture is lowered to ~50˚C, allowing the sequencing primer to bind to a complementary sequence on one strand of the COI amplicon. The sequencing primer that was added to the forward sequencing reaction binds or anneals to a complementary sequence on the antisense strand according to the base pairing rules. The sequencing primer that was added to the reverse sequencing reaction binds or anneals to a complementary sequence on the sense strand.
3. During the elongation step, each reaction mixture is raised to ~60˚C. At this temperature, a heat-stable DNA polymerase finds the 3’ end of the sequencing primer and begins joining nucleotides that are complementary to those present in the template strand. For the forward sequencing reaction, the DNA polymerase joins nucleotides that are complementary to those in the antisense strand. For the reverse sequencing reaction, the DNA polymerase joins nucleotides that are complementary to those in the sense strand.
   During this step of the sequencing reaction, the DNA polymerase cannot distinguish between dNTPs and ddNTPs present in the reaction mixture. Because a higher proportion of dNTPs were added to each sequencing reaction mixture, they are more likely to be incorporated into the growing DNA chain. However, when a ddNTP lacking a 3’ –OH is incorporated, DNA synthesis stops as no new nucleotides can be added to the growing chain.
   The denaturation, annealing, and elongation steps are repeated multiple times, thereby ensuring that at the conclusion of each sequencing reaction, single-stranded DNA fragments of every possible length are generated. Importantly, each fragment terminates with one of the four ddNTPs, which are labeled with a different fluorescent tag.

Upon completion of each sequencing reaction, the fluorescently labeled DNA fragments are separated according to size using capillary electrophoresis. As the DNA fragments migrate from smallest to largest through the capillary, they pass through a laser, which excites the fluorescent ddNTP at the terminal end of each fragment. DNA fragments terminated by ddATP emit green light DNA fragments terminated by ddTTP emit red light DNA fragments terminated by ddGTP emit yellow light DNA fragments terminated by ddCTP emit blue light The light emitted from fluorescent labeled DNA fragments is detected by the sequencer and represented as a continuous series of colored peaks in an electropherogram or trace file. The peak from the smallest fluorescently labeled DNA fragment is represented first in the trace file, whereas the peak from the largest fragment is represented last. The information contained in a trace file will be discussed in greater detail below.