|AUTOMATED DNA SEQUENCING|
Automated deoxyribonucleic acid (DNA) sequencing reduces the volume of low-level radioactive waste generated on campus, while providing a suitable alternative to manual DNA sequencing. Traditional methods of manual DNA sequencing utilize radioactive isotopes to label the DNA. Automated DNA sequencing utilizes fluorescent tracers instead of radioisotopes to label the DNA, thereby eliminating or significantly reducing the use of radioactive materials in some research laboratories.
40 CFR Parts 260-268.
10 CFR 20 Subpart K.
Overview of Procedure
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.
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.
& Health Precautions/Personal Protective Equipment
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.
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.