?/SUP> gels* for the LI-COR system are provided polymerized and are ready to load in less than 30 minutes. Use the CastAway gels with LI-COR抯 IR2 Automated DNA Analysis System to obtain more than 16,000 bases of sequence data or to analyze 1,440 genotypes per gel.
DNA sequence analysis remains an essential technology for elucidating gene structure and function. For more than a decade, automated methods have been developed to improve the throughput and accuracy of DNA sequencing and genotyping.1,2,3,4 In general, the technology involves the real-time detection of fluorescently labeled DNA fragments as they move through a portion of an electrophoresis gel that is irradiated by a laser. The fluorescence emission is collected by a detector located at a fixed distance from the loading wells. The resultant signal produces a band pattern, which correlates to a DNA sequence or to a specific DNA fragment size for genotyping applications.
One limitation of automated DNA sequencers is the time required to cast gels used to separate DNA fragments electrophoretically. Hence, LI-COR and Stratagene adapted the CastAway precast sequencing gels5 for use on the Model 4200 (IR2) Automated DNA Analysis System.6,7 By using precast gels on an infrared fluorescence automated DNA sequencer, convenience and throughput in DNA sequence analysis are improved.
Precast Gels
Figure 1
Pouring a slab gel the conventional way can take more than 2 hours: assemble the gel apparatus, mix reagents, degas the gel solution, pour the gel, and complete gel polymerization. In contrast, Stratagene抯 CastAway gels are provided polymerized and ready to load in less than 30 minutes (Figure 1).
The gel apparatus and precast gel are easily assembled in four steps (Figure 1). First, remove the CastAway gel from its packaging and allow it to equilibrate to room temperature. Second, rinse the front and back glass plates with distilled water and dry with lint-free wipes. Third, tighten the left and right precast gel rails and the upper buffer tank into position, and the gel assembly is complete. Finally, install the gel apparatus on the LI-COR instrument and carry out a 20 minute pre-electrophoresis prior to loading the gel.
Focusing and Electrophoresis
The LI-COR IR2 Automated DNA Analyzer uses a scanning infrared fluorescence microscope to detect the electrophoretic movement of fluorescently labeled DNA molecules in real time. The system is a two-dye infrared automated DNA sequencer that can detect the products of two sequencing reactions in parallel by generating two separate gel images, each corresponding to the fluorescence emission of a different infrared dye (700 nm channel for IRD 700; 800 nm channel for IRD 800).
The microscope consists of one or two laser diodes for providing dye excitation (wavelength at 685 nm and 785 nm) and one or two avalanche photodiodes for detecting the fluorescence emission.6,7 By using infrared radiation, the background fluorescence caused by the soda lime glass of the precast gel is greatly reduced. Moreover, variations in precast gel thickness can be accommodated by autofocusing the fluorescence microscope to assure optimal signal-to-noise ratios for a given gel configuration (Figure 2).
Figure 2
DNA reaction products are heat denatured and loaded on the CastAway gel using a 48- or 64-well sharktooth comb. To separate DNA fragments produced in a sequencing reaction, electrophoresis is carried out at a constant 25 W (ca. 50 V/cm and 18 to 22 mA) (Figure 3). To obtain banding patterns of short tandem repeat polymorphisms (STRPs), fluorescently labeled PCR products are separated using electric field conditions of 1000 V, 45 mA, and 45 W (Figure 4). During electrophoresis, the precast gel is maintained at 45篊 by a thermostatically controlled heater plate that is in contact with the rear glass plate. After electrophoresis, precast gels are discarded, thus eliminating the need to clean glass plates.
Figure 3
DNA Sequencing and Genotyping Applications
The Model 4200 (IR2) Automated DNA Analysis System was used in conjunction with the CastAway precast gel to obtain sequence from M13mp19 single-stranded DNA using the M13 (-29) forward primer (Figure 3). The autosequencer called 600 bases with 99.7% accuracy, and data were collected in 2.5 hours (from time of load) using the aforementioned electrophoresis conditions. Read lengths greater than 500 bases are typically achieved for sequence ladders generated on precast gels and analyzed in either the system抯 700- or 800-nm channel; this compares to read lengths obtained with manually poured gels (data not shown). The total base number per precast gel is greater than 16,000 when the gel is maximally loaded (i.e., 32 sequencing samples).
For genetic analysis, STRP banding patterns were obtained using a precast gel on the automated infrared DNA analyzer. In Figure 4, four STRP loci are detected in each lane (four multiplexed reactions per channel). Expect 1,440 genotypes (60 lanes x 4 loci x 2 channels x 3 loads) for a typical throughput per precast gel, which is equivalent to manually poured gels. Additionally, the precast gel can be reloaded three times (Figure 4), making fluorescence STRP analysis cost effective as well.
Figure 4
Conclusions
Use Stratagene抯 CastAway gels for the LI-COR system on the IR2 DNA Analysis System for automated DNA sequencing and genotyping applications. CastAway precast gels are made with soda-lime float glass plates, which produce minimal background fluorescence with the system抯 infrared technology. Therefore, it is unlikely these precast gels could be used with visible fluorescence automated DNA sequencers, such as the ABI PRISM?/SUP> 377, since the system sensitivity would be lower due to significant emission of background fluorescence by the soda-lime plates. Further, CastAway gels compare favorably to the standard LI-COR manually poured 25-cm gel梬ith respect to electrophoresis run time, band morphology, band resolution, and signal strength梐nd both gel types produce a similar sequence read length and accuracy.
