Dye Labeling

Dye labeling is typically used for sample identification in the automatic DNA sequencers. The properties of a dye shall satisfy the following major requirements: 1) Dye structure shall allow attachment to a primer or ddNTP with a reasonable yield. 2) Absorption spectrum shall match the laser wavelength. 3) Extinction coefficient and quantum yield shall be high enough to provide acceptable sensitivity. 4) Dyes shall be chemically and thermally stable in order to withstand cycle sequencing conditions 5) Dyes shall be photo stable. 6) Mobility of the DNA fragments labeled with different dyes shall be equalized, which can be achieved by optimization of the labeling

process.1 ]

Both emission and absorption spectra of the organic dyes employed in DNA labeling are broad (typically 2050 nm). This relaxes the requirement for spectral content of the excitation light.

Overlapping absorption spectra of several dyes used for labeling allows excitation of all these dyes with a single laser source. However, efficiency of excitation drops off quickly as the laser wavelength deviates from the absorption maximum of the dye and results in a significant drop in sensitivity. To avoid these losses, energy transfer (ET) dyes were introduced.[9]

In the case of multicolor sequencers (when two or more dyes are used for DNA labeling), collected radiation must be spectrally separated by means of a diffractive element or a set of interference filters and is registered by separate PMTs or by a CCD. Alternatively, a single PMT can be used with a set of replaceable filters (a ''color wheel''). A more sophisticated detection method used for sensitivity increase is based on single-photon detection.[10]

A complication arises because the emission spectra of the dyes used for labeling different samples overlap and color cross-talk correction of the collected data is required. Improper compensation of the cross-talk results in false peaks in the processed data.

Both visible and near IR dyes can be used for DNA labeling in automated DNA sequencers. The advantage of using red and near IR dyes is lower background

Fig. 1 A typical result of base calling obtained with an automated DNA sequencer. The Ml3 sequencing by ultrathin slab gel electrophoresis. The signal obtained with the Long-Read Tower™ sequencer has been base called with GeneObjects software. (From Ref. [7].) The accuracy of base calling is 98.5 % up to 500 bases. Similar results are typically produced with other types of automated DNA sequencers, including CE instruments.

Fig. 1 A typical result of base calling obtained with an automated DNA sequencer. The Ml3 sequencing by ultrathin slab gel electrophoresis. The signal obtained with the Long-Read Tower™ sequencer has been base called with GeneObjects software. (From Ref. [7].) The accuracy of base calling is 98.5 % up to 500 bases. Similar results are typically produced with other types of automated DNA sequencers, including CE instruments.

fluorescence and decreased laser light scattering at longer wavelengths. This results in better sensitivity. IR dyes can be excited by laser diodes (LD) resulting in a reduction of instrument design complexity.1-6'7-1

Dye labels can be covalently linked either to a primer or to each of four types of ddNTPs.[11] In the last case' all four sequencing reactions can be carried out in one tube instead of the four used in primer-labeled chemistry. However, dye-labeled terminators may introduce significant variability in the peak intensity resulting in decreased base calling accuracy and reliability. For clinical applications dye primer chemistry may be preferred because of the higher reliability of the results. Terminator chemistry provides more flexibility, which may be advantageous for the research laboratories.

In some automated sequencers less than four colors are used for DNA labeling. For example, a two-dye approach is used in the Long-Read Tower™ and LICOR sequencers. The sample is prepared by running two sequencing reactions in the same tube with differently labeled forward and reverse primers. This approach enables the increase of the read length, accuracy, and reliability of base calling.

TYPES OF DNA SEQUENCERS Slab Gel Sequencers

Initially, automated DNA sequencers were based on slab gel electrophoresis with low throughput and automation limited to data collection and analysis. Gel filling and sample loading are done manually. The support glass plates require cleaning after each run. Poor dissipation of heat generated in a slab gel through relatively thick glass plates limits the electric field strength and prevents short run times. Both of these problems are eliminated in the disposable ultrathin slab gels (50 mm thick), which can withstand higher electric field strength, E (100 V/cm), resulting in shorter run times.[7]

Capillary Electrophoresis Sequencers

Capillary electrophoresis (CE) DNA sequencers were introduced to increase analysis throughput. This is a direct result of high electric fields (100-250 V/cm) and multiple capillaries (up to 384 capillaries). Both sieving matrix replacement and sample loading can be automated. The small diameter of capillaries (internal diameter is 50-100 mm) allows for good heat dissipation. Capillaries having smaller diameters (2-10 mm) are not practical as they do not provide enough signal intensity, are difficult to fill with acrylamide, and do not provide reproducible performance.1-12-1 The ability to use replaceable sieving matrices makes CE systems practical as repetitive runs are possible with the same capillary up to 200 times or more.

Electra-kinetic sample loading allows high automation of CE sequencers. But optimization of loading conditions and sample purification is required. These are critical for reducing variability of peak intensity, suppressing decay in gel current and bubble formation, increasing capillary lifetime. Salt concentration, ratio of labeled DNA to unlabeled DNA, and total volume of loaded sample all affect the success of CE analysis.

Typical read length in CE DNA sequencers is 400600 bases in several hours, but can be as high as 1100 bases.[13] Run conditions can be optimized for faster runs, if necessary, by reducing separation lengths, decreasing polymer concentration, increasing voltage or/ and gel temperature.

Examples of commercial automated DNA sequencers are shown in Table 1.

A variety of automated DNA sequencers is available, providing similar performance characteristics but

Table 1 Automated DNA

sequencers

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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