Automated DNA Cycle Sequencing Combines PCR and Sequencing

AUTOMATED DNA CYCLE SEQUENCING COMBINES PCR AND SEQUENCING

Automated DNA sequencing uses a PCR-type reaction to sequence DNA. This is an improved way to sequence DNA because of its speed and because it can be analyzed by computer rather than a person. In PCR sequencing, or cycle sequencing, the template DNA with unknown sequence is amplified by Taq polymerase rather than Klenow polymerase. As for DNA polymerase I, this Taq DNA polymerase was modified to remove its proofreading ability. Cycle sequencing reaction mixtures includes all four deoxynucleotides, all four dideoxynucleotides, a single primer, template DNA, and Taq polymerase. Unlike regular sequencing, each dideoxynucleotide is labeled with a fluorescent tag of a different color; therefore, all four dideoxynucleotides are in the same reaction mix.

The samples are amplified in a thermocycler. First, the template DNA is denatured at a high temperature, then the temperature is lowered to anneal the primer, finally the temperature is raised to 70°C, the optimal temperature for Taq polymerase to make DNA copies of the template. During polymerization, dideoxynucleotides are incorporated and cause chain termination, just as with standard sequencing. The ratio of dideoxynucleotides to deoxynucleotides is adjusted to ensure that some fragments stop at each G, A, T, or C. After Taq polymerase makes thousands of copies of the template, each stopping at a different nucleotide, the entire mixture is run in one lane of a sequencing gel (Fig. 4.21). Bands of four different colors are seen, corresponding to the four fluorescently labeled dideoxynucleotides and hence the four bases.

Cycle sequencing has some advantage over regular sequencing. During regular sequencing reactions, the temperatures are lower; therefore, the template DNA is more likely to fold back on itself into different secondary structures. This is especially true for double-stranded DNA templates, which can reanneal before Klenow polymerase has a chance to work. During cycle sequencing, each round brings the temperature to 95°C, which destroys any secondary structures or double-stranded regions. Another advantage of cycle sequencing is to control primer hybridization. Some primers do not work well with regular sequencing reactions because they bind to closely related sequences. During cycle sequencing, the primer annealing temperature is controlled and can be set quite high in order to combat nonspecific binding. Finally, cycle sequencing requires much less template DNA than regular sequencing; therefore, sequencing can be done from smaller samples.

Another advance in sequencing has been the detection system. Traditionally, researchers have used radioactively labeled nucleotides, followed by autoradiography to detect the bands. A major improvement has been to use fluorescently labeled dideoxynucleotides; therefore, the terminal nucleotide of each DNA strand will fluoresce. Different fluorescent tags can be used for each of the four dideoxynucleotides, so that the whole reaction can be done in one tube. Automatic DNA sequencers detect each of the fluorescent tags and record the sequence of bases (Fig. 4.22). Much like regular sequencing, the different fragments are run on a gel, but instead of four lanes, the entire mix is all in the same lane. Some automatic DNA sequencers can read up to 96 samples side-by-side on each gel. At the bottom of each lane is a fluorescent activator, which emits light to excite the fluorescent dyes. On the other side is the detector, which reads the wavelength of light that the fluorescent dye emits. As each fragment passes the detector, it measures the wavelength and records the data as a peak on a graph. For each fluorescent dye, a peak is recorded and assigned to the appropriate base. An attached computer records and compiles the data into the DNA sequence.

Automated sequencing has a large startup cost because the sequence analyzer is quite expensive, but they run multiple samples at one time, and thus the cost per sample is quite low. Many universities and companies have a centralized facility that does the sequencing for all the researchers. In fact, sequencing has become so automated that many researchers just send their template DNA and primers to a company that specializes in sequencing.