DNA Sequencing

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DNA sequencing has proved to be an invaluable tool across many disciplines of molecular biology, including basic research, forensics, and diagnostics. For the purposes of molecular diagnostics, sequencing combines the sensitivity of PCR with unparalleled accuracy of identification, and  has become the "gold standard" to detect genetic anomalies.  Viracor-IBT has utilized sequencing technology for a number of research and diagnostic applications within infectious disease and immunological testing. Several of our assays utilize DNA sequencing for the assessment of mutations which confer antiviral drug resistance. Applying DNA sequencing to infectious disease and immunological diagnostics has enabled Viracor-IBT to offer diagnostic tools that enable earlier diagnosis and treatment of many critical conditions.

In 1974, a group headed by Frederick Sanger developed the principles of DNA sequencing for a process now known as Sanger dideoxy sequencing. More recently, refinement and extension of the Sanger method has led to the application of more efficient automated, fluorescence-based, cycle sequencing systems for mainstream applications. Like Sanger sequencing, fluorescence-based cycle sequencing exploits the ability of DNA polymerase to incorporate 2', 3'-dideoxynucleotides, nucleotide base analogs that lack the 3'-hydroxyl group essential in phosphodiester bond formation. Much like traditional PCR reactions, sequencing requires a DNA template, a sequencing primer, a thermal stable DNA polymerase, nucleotides (dNTPs), dideoxynucleotides (ddNTPs), and buffer. Unlike Sanger's original method, which uses radioactive material, cycle sequencing uses fluorescent dyes to label the extension products. These components are combined in a reaction that is subjected to cycles of annealing, extension, and denaturation in a thermal cycler. The cycle sequencing reaction is directed by highly modified, thermally stable DNA polymerases which allow incorporation of dideoxynucleotides. These modified DNA polymerases are also formulated with a pyrophosphatase to prevent reversal of the polymerization reaction (pyrophosphorolysis). The thermal cycling process creates and amplifies extension products that are terminated by one of the four dideoxy nucleotides (Figure 1). The ratio of deoxynucleotides to dideoxynucleotides is optimized to produce a balanced population of long and short extension products. With dye terminator chemistry, each of the four dideoxynucleotide terminators is tagged with a different fluorescent dye (Figure 2). Once thermal cycling has been completed, the reaction products are subjected to electrophoresis for the purposes of separation and identification. Historically, DNA sequencing products were separated using polyacrylamide gels that were manually poured between two glass plates. Capillary electrophoresis, which utilizes a denaturing flowable polymer, has largely replaced the use of gel separation techniques, providing for gains in workflow, throughput, and ease of use.

Figure 1

Four dideoxy nucleotides

Figure 2

 

During capillary electrophoresis, a high voltage charge applied to the buffered sequencing reaction forces the negatively charged fragments into the capillaries. The fluorescently labeled extension products are separated by size based on their total charge and move across the path of a laser beam, which causes the dyes to fluoresce. An optical detection device on a genetic analyzer detects the fluorescence, and data collection software converts the fluorescence signal to sequence data. Because each dye emits light at a different wavelength when excited by the laser, all four colors, and all four bases, can be detected and distinguished in a single capillary.

Analyzed sample data is displayed as an electropherogram, a sequence of peaks in four colors. Each color represents the base called for that peak (Figure 3).

Figure 3

Electropherogram

Once sequence data has been procured and assessed for quality, it may be utilized for a number of different applications, including de novo sequencing of genomes, detection of genomic variants and mutations, and biological identification. For mutation assessment and biological identification, the generated sequence is aligned and compared with known sequences in curated sequence databases. For example, Viracor-IBT utilizes the SmartGene® Bacteria IDNS database for identification of bacterial infectious disease pathogens based on generated 16s rDNA sequences.

References


Sanger, F., Donelson, J.E., Coulson, A.R., Kösse,l H., Fischer D. 1974.
Determination of a nucleotide sequence in bacteriophage f1 DNA by primed
synthesis with DNA polymerase. J Mol Biol. 90(2): 315-33.

Applied Biosystems. 2009. DNA Sequencing by Capillary Electrophoresis Applied Biosystems Chemistry Guide, 2nd ed., 310 pp.