Automated DNA Sequencing

Applicable Regulations
40 CFR Parts 260-268.

10 CFR 20 Subpart K.

Overview of Procedure
DNA is the main carrier of genetic information in living organisms. DNA molecules are extremely long, large, and consist of repeating nucleotides. Nucleotides are the bases of DNA and consist of adenine (A), thymine (T), guanine (G), and cytosine (C). The structure of a DNA molecule is double stranded, consisting of two DNA strands wound around each other to form a double helix. The nucleotides of the two strands are complementary to each other such that adenine cross-links with thymine (A-T), and guanine cross-links with cytosine (G-C). The goal of DNA sequencing is to determine the order of bases for a specific piece of DNA.

DNA sequencing is achieved by utilizing labeled nucleotides for incorporation into a copy of a piece of DNA. The DNA sequence can then be derived by the positions of the labeled nucleotides. First, the DNA segment to be copied, called the template DNA, is separated into two strands by heating. An enzyme is used to make complementary copies of the individual strands with the labeled nucleotides. The DNA segments are then separated according to length by electrophoresis in a polyacrylamide gel. Electrophoresis is the movement of electrically charged particles through a porous substance (polyacrylamide gel) under the influence of an electric field provided by a high voltage unit. Once the DNA is separated, several different techniques are available to allow for analysis of the DNA sequence.

Traditional methods of manual DNA sequencing utilize radioactive isotopes such as phosphorous-32, sulfur-35, and phosphorous-33, incorporated into specific nucleotides (A,C,T,G). Radioactive labeled nucleotides allow for reading the sequence by a technique known as autoradiography. The gel that contains the separated DNA segments is exposed to X-ray film for a period of time. The radiation causes dark spots on the film to indicate its location. Next, the film is developed to reveal the pattern of the labeled nucleotides. Since a process does not exist to discriminate the different nucleotides by the spots on the film, each labeled nucleotide must have its own lane on the gel. Therefore, four individual lanes are required for manual sequencing in order to determine the full DNA sequence. An individual must interpret the results of this process and typically the results are entered into a computer for storage and linking to other results.

Waste Minimization Procedure
Automated DNA sequencing utilizes a fluorescent dye to label the nucleotides instead of a radioactive isotope. The fluorescent dye is not an environmentally hazardous chemical and has no special handling or disposal requirements. Instead of using X-ray film to read the sequence, a laser is used to stimulate the fluorescent dye. The fluorescent emissions are collected on a charge coupled device that is able to determine he wavelength. The Perkin-Elmer Applied Biosystems (ABI) DNA sequencers are designed to discriminate all four fluorescent dye wavelengths simultaneously, which allows for complete DNA sequencing in one lane on the gel.

Varying degrees of automation are also available. For full automation, all that is required is to load a sample tray with template DNA; the equipment performs the labeling and analysis. The other option is to perform the labeling reactions with fluorescent dyes, load the samples onto a gel, and place the gel into the DNA sequencer. The equipment performs the separation and analysis. The system automatically identifies the nucleotide sequence and saves the information on the computer. Thus, only a review of the data is necessary to ensure no anomalies were misidentified by the computer.

Known Limitations
The greatest obstacle to researchers when converting from manual to automatic DNA sequencing is being required to learn the use of computer software necessary to interpret the results.

Safety & Health Precautions/Personal Protective Equipment
Follow all applicable safety and health protocols and regulations as established by your institution.

Benefits
Automated DNA sequencing equipment can eliminate the need for radioactive isotopes to label DNA, thereby reducing the volume of low-level radioactive waste generated on campus.

As a general approximation, one template of manual DNA sequencing will produce 83 mL of liquid waste and 0.167 gallon of solid waste. As a result, every 45 templates processed by automated DNA sequencing reduces the amount of manual DNA sequencing. The time saved is due to not having to perform autoradiography or associated tasks required for working with radioactive materials such as radiation surveys, inventory/disposal documentation, etc.

Finally, automated DNA sequencing provides more reliable research results than manual DNA sequencing, thus maintaining the integrity of the research.

Disadvantages

None known.

Project Related Costs
Material costs for automated and manual DNA sequencing have some key differences. In both methods, reagent costs are comparable, but the manual method requires four lanes per template versus one lane in the automated method. In addition, the manual method utilizes radioactive labels, thereby generating low-level radioactive waste with an associated disposal cost. The manual method also utilizes a special x-ray film to perform autoradiography with associated developing costs. In general, the total cost of material for one gel using the automated method is approximately $180, compared to $360 for the manual method.

Automated DNA sequencing equipment is offered in a variety of models and options.

One model starts at $120,000 for all necessary equipment to perform analysis only. Additional accessories are available and range from $50 to $2,000. Optional software is available for an additional $10,000.

The payback period will vary depending upon the cost of the equipment purchased and the volume of DNA sequencing performed. One estimate found that with a standard model that performs analysis only, the payback period could range from four to sixteen months, depending upon the frequency of use. For fully automated equipment, the payback period could be as much as two years. It is important to note that factors that tend to increase the costs of manual DNA sequencing (e.g., the long operation time; associated tasks and personal protection required for working with radioactive material) are not included in the costs of automated DNA sequencing.