Cycle Sequencing Protocol
Step 1: A DNA sequence of interest is first
amplified using the polymerase chain reaction (PCR).
to reaction tube:
DNA sequence of interest
DNA polymerase (a heat-resistant enzyme that catalyzes the addition
primer (a short single-stranded DNA sequence that base pairs to the
DNA sequence of interest and serves as a site from which to add complementary
supply of the four deoxynucleotides
to separate DNA strands
to allow primers to bind
- Taq DNA polymerase extends the 3'
end of each primer to produce double-stranded DNA molecules
for 20 cycles.
Step 2: The amplified DNA strands are subjected
to the DNA sequencing reactions.
- Add to reaction tubes:
DNA strand from Step 1
(human-made nucleotides that can not
form covalent bonds to another nucleotide and are labeled with a fluorescent
dye. The addition of a dideoxynucleotide terminates the growing DNA
through 20 30 cycles of heating and cooling.
Step 3: The results of the sequencing reactions are separated
by capillary gel electrophoresis and visualized using fluorescent dyes.
the contents of the sequencing reactions into capillary tubes. We use capillary tubes made of fused silica
(glass) and coated on the outside with polyimide (for strength). The
tubes have an internal diameter of 100 micrometers and are 100 cm in
length. The tubes contain a gel solution which serves as a sieving matrix.
Each of our electrophoresis machines holds 96 capillary tubes.
DNA strands migrate, and therefore separate from each other, according
to size. The smallest strand travels the fastest and is detected first.
laser beam directed at the bottom of the capillary tubes excites the
fluorescent-labeled dideoxynucleotide which is positioned at the end
of the DNA strand.
photodetector measures the light emitted by each dideoxynucleotide
as it passes through the laser beam.
computer records each wavelength of light and generates an electropherogram
with colored peaks representing each wavelength. The terminal base
(the dideoxynucleotides) of the shortest fragment is the first base
in the electropherogram. The colors of each base are listed below:
- Guanine (G) yellow (the tracings on the chart paper use black
instead of yellow)
- Cytosine (C) blue
- Adenine (A) green
- Thymine (T) red
- Unknown base = N