TY - JOUR
AU - Dovichi, Norman J.
AB - Abstract A five-capillary system has been developed for DNA sequencing and analysis. The post-column fluorescence detector is based on a sheath-flow cuvette. The instrument provides uniform and continuous illumination of the samples. The cuvette virtually eliminates cross-talk in the fluorescence signal between capillaries. Discrete single-photon counting avalanche photodiodes provide high efficiency light detection. The instrument has detection limits (3σ) of 130 ± 30 fluorescein molecules injected onto each capillary. Over 650 bases of sequence at 98.8% accuracy were generated in 100 min at 50°C from M13mp18. Separation and detection of short tandem repeats proved efficient and accurate with the use of internal standards for direct comparison of migration times between capillaries. Introduction Large-scale DNA sequencing projects require instruments that generate high throughput and high sequencing accuracy at low cost (1). Capillary electrophoresis provides low-cost, easily automated and rapid DNA sequencing (2–15). The first multiple-capillary instrument was reported in 1990. Zagursky modified a commercial DuPont Genesis 2000 sequencer to operate with 500-µm ID capillaries (16). In that instrument, an argon ion laser beam was scanned across the capillary array. The instrument operated at 50 V cm−1; 9.5 h were required to separate fragments 500 bases in length. Sequencing accuracy was <97% for fragments ranging from 29 to 512 bases in length. Mathies reported a similar scanning instrument to image a capillary array (17). That instrument operated with 100-µm ID capillaries and produced sequencing information up to 320 bases in length. Duty cycle is an important parameter in specifying a detector's performance. In scanning systems, the optical system probes each capillary in sequence. Duty cycle is the fraction of time that a sample is illuminated. Duty cycle is important because DNA fragments migrate from the capillary undetected during the period when a capillary is not illuminated. In scanning systems, the duty cycle decreases in proportion to the number of capillaries. In contrast, several systems have been developed to continually monitor fluorescence from a capillary array; these systems inherently have a much higher duty cycle than scanning systems. Yeung's group reported a multiple-capillary DNA sequencer in which a ribbon of capillaries was illuminated with a line-focused laser beam. Fluorescence was collected at right angles and imaged onto a CCD camera. The use of the CCD camera ensured that all capillaries were monitored simultaneously (18,19). Similarly, an eight-capillary DNA sequencer was reported based on the use of individual fiber-optics to deliver a laser beam to each capillary (20). A discrete laser beam is required to excite fluorescence from each sample. A second set of fibers transmits fluorescence to an imaging spectrograph and CCD detector. The sequence from 400 to 450 bases was generated in 1 h. These designs are relatively inefficient in their use of excitation light. For example, if 10 mW of light is required to excite fluorescence from each capillary, then 1 W of light is required to excite fluorescence from 100 capillaries. In a further improvement, both Yeung and Kambara have reported a capillary array approach with side-illumination, on-column fluorescence detection (21,22). These instruments provide continual illumination of the samples with one laser beam, providing a much higher duty cycle compared to a scanning instrument and requiring a much lower laser power than used in the line-focused or fiber-optic excited systems. The on-column detection scheme is useful for small numbers of capillaries but appears to be difficult to scale to larger arrays. Kambara showed another approach by applying post-column fluorescence detection in a sheath-flow cuvette (23,24). Sequencing length of 303 bases was achieved in 111 min. Our research group reported the first capillary electrophoresis instrument based on post-column fluorescence detection in a sheath-flow cuvette (25). These detectors, borrowed from the optical chamber used in flow cytometry, provide very low background and very high sensitivity fluorescence detection, which allows the detection of individual fluorescent molecules migrating from a capillary electrophoresis column (26). We first reported the use of the sheath-flow cuvette for separation of single-base termination DNA sequencing fragments by capillary electrophoresis in 1990 (6). The system was expanded to four-color operation in 1991 (9). We reported a single-capillary electrophoresis instrument that operates at elevated temperatures with non-crosslinked polyacrylamide (15). The 0%C polymer has low viscosity and may be pumped from the capillary and replaced with fresh material after each run. Sequencing fragments over 640 baseswere separated in 2 h at an electric field of 150 V cm−1 and at a temperature of 60°C. This report was the first description of high temperature separation of DNA sequencing fragments in non-crosslinked polymer. The high temperature operation increased sequencing rate, decreased compression, and increased the sequencing read length compared to room temperature sequencing. Figure 1 View largeDownload slide Four-spectral-channel laser-induced fluorescence detection. The sector wheel alternately transmits the two laser beams and the filter wheel synchronizes the transmission of fluorescence in four bands chosen to match the emission spectra of the four dyes. Figure 1 View largeDownload slide Four-spectral-channel laser-induced fluorescence detection. The sector wheel alternately transmits the two laser beams and the filter wheel synchronizes the transmission of fluorescence in four bands chosen to match the emission spectra of the four dyes. In this paper, we report a five-capillary version of the high-efficiency electrophoresis system (27). The instrument provides high sensitivity, which is important for the detection of the very small amount of sample loaded onto the capillary. The instrument also can operate at high temperature, which minimizes the formation of compressions, and produces faster separations and longer read-length (15). This instrument fills a niche between the single-capillary Applied Biosystems 310 and the larger-scale Applied Biosystems 377 slab-gel system and the Applied Biosystems 3700 capillary array instrument, and should be of interest to small-scale sequencing laboratories. Materials and Methods Instrumental design The overall scheme for the instrument is shown in Figure 1 (27). Two lasers, a 1-mW helium-neon laser operating in the green at 543.5 nm and a 4-mW argon ion laser operating in the blue at 488 nm, were alternately chopped with a sector wheel to provide sequential excitation. The beams were recombined with a dichroic beam splitter and focused with a 1× microscope objective into the locally designed multiple-capillary sheath-flow cuvette. Figure 2 View largeDownload slide The sheath-flow cuvette fluorescence detection chamber for an array of five capillaries. The chamber is tapered, being 50 µm wider at the top than at the bottom. A single laser beam is used to illuminate fluorescence from the five sample streams isolated by the sheath flow fluid. Figure 2 View largeDownload slide The sheath-flow cuvette fluorescence detection chamber for an array of five capillaries. The chamber is tapered, being 50 µm wider at the top than at the bottom. A single laser beam is used to illuminate fluorescence from the five sample streams isolated by the sheath flow fluid. The rectangular sheath-flow cuvette (Fig. 2) was constructed from high quality quartz by NSG Precision Cells. The cuvette had a 150 µm × 750 µm inner chamber, four 1-mm thick windows and a height of 2 cm. The two narrow walls of the chamber were tapered so that their spacing was 50 µm wider at the top than at the bottom. The taper forced the capillaries to be squeezed together as they were inserted into the cuvette. Sheath fluid was pumped through the interstitial space between the capillaries. Either 10 mM borate (for free-zone electrophoresis) or 1× TBE (for gel electrophoresis) was used as the sheath fluid. This buffer drew the sample from the capillaries, creating a set of independent sample streams, one sample stream centered beneath each capillary. Fluorescence from each sample stream was excited simultaneously by the laser beam that was focused ∼100 µm below the ends of the capillaries. A microscope objective (20× and 0.5 NA) collected fluorescence. A filter wheel was located between the collection optic and the detectors. This filter wheel was equipped with four 1- inch-diameter interference filters. For sequencing, the bandpass of the filters was centered at 540, 560, 580 and 610 nm with a 10-nm bandwidth. For short tandem repeat analysis, the bandpass was centered at 530, 545, 560 and 580 nm. The sector wheel, which alternately transmitted the blue and green laser beams, was synchronized with the filter wheel by use of stepper motors driven by a common controller. Excitation at 488 nm was synchronized with detection at the two shorter wavelength filters. Excitation at 543.5 nm was synchronized with detection at the two longer wavelengths. The filter wheel was equipped with sensors to signal the identity of the filter in the optical path. It was not possible to image the fluorescence directly onto the photodetectors; their relatively large size was incompatible with the spacing of the fluorescence image generated by the collection optic. Instead, the fluorescence was imaged onto a set of five gradient refractive index (GRIN) lenses. These GRIN lenses were 1.8-mm diameter, 0.25 pitch and arranged at 3-mm center spacing; there was one GRIN lens per fluorescent spot. Each GRIN lens coupled the fluorescence to a fiber optic, which was pig-tailed to an avalanche photodiode (APD). These photodiodes operated in the single-photon-counting mode and provided low dark count, high quantum efficiency photodetection. The output of the single-photon-counting modules consisted of a train of pulses. The pulse train was converted to a voltage with a frequency-to-voltage converter, which was monitored with an analog-to-digital board. A Macintosh Quadra 700 was used for data acquisition. Capillary electrophoresis A 30-kV power supply was used to drive electrophoresis. The samples or running buffers were encased in a Plexiglas box equipped with a safety interlock. The capillaries were held in a locally constructed heater based on Peltier devices and a proportional temperature controller, which held the temperature of the capillaries with an accuracy of ± 0.5°C. The sheath flow was provided either by a precision syringe pump or by simply siphoning with the height difference of 5–2 cm between sheath flow reservoir and waste container. Sheath flow rate was typically 0.3 ml h−1. Reagents and solutions Fluorescein was a high purity standard from Molecular Probes. Borax, boric acid and EDTA were analytical-reagent grade. Tris base and urea were ultra pure reagents. Acrylamide and N,N,N′,N′-tetramethylethylenediamine (TEMED) were electrophoresis-purity reagents. Ammonium persulphate was ultra pure electrophoresis grade. [γ-(methacryloxy)propyl]trimethoxysilane was reagent grade. A stock borate solution was prepared by dissolving 0.478 g of borax in 50 ml water. It was diluted to a final concentration of 10 mM borate (pH 9.2). A stock solution of ∼1 mM fluorescein was made by dissolving fluorescein in ethanol. A series of four concentrations of fluorescein were prepared from 2 × 10−11 to 2 × 10−12 M by diluting the stock fluorescein solution with 10 mM borate. A 1% [γ-(methacryloxy)propyl]trimethoxysilane solution was prepared by adding 10 µl of [γ-(methacryloxy)propyl]trimethoxysilane in 990 µl 95% ethanol-5% water solution (pH 4.8). A stock 10× TBE (pH 8.3) solution was prepared by dissolving 108 g Tris, 55 g boric acid and 40ml of 0.5MEDTAin water to a final volume of 1 liter. Capillary polymer preparation We coated the capillaries using a method reported earlier (15). By using a water aspirator, the capillaries were first filled with 1% [γ-(methacryloxy)propyl]trimethoxysilane solution; after 20 min, the silane solution was replaced with degassed 5% acrylamide, 7 M urea, 0.07% (w/v) ammonium persulphate and 0.07% (v/v) TEMED solution prepared in a TBE buffer (1× TBE final). The polymerization reaction was allowed to proceed overnight. The 5%T polyacrylamide solution-filled capillary was subjected to a 30-min pre-run before sample injection. The four-color sequencing reaction products were separated using a non-crosslinked dimethylacrylamide polymer solution. The polymer was prepared from 6% dimethylacrylamide, 7 M urea, 0.07% (w/v) ammonium persulphate and 0.07% (v/v) TEMED solution prepared in a TBE buffer (1× TBE final) under an argon atmosphere. The polymerized solution was loaded into a 10-ml syringe and stored at 4°C before use. The polymerized solution was injected into the capillary using a homemade manifold system. The separation was performed at 50°C, the capillary length was 60 cm and the electric field was 185 V cm−1. Sequencing sample preparation and injection All sequencing templates were M13mp18. The four-color sample was a cycle-sequencing product (15). A Sequitherm Long-Read Cycle Sequencing kit was used for cycle sequencing with fluorescently labeled primers. Cycle sequencing reactions were carried out according to the recommended protocols of the manufacturer. Cycle sequencing was performed using 30 cycles on a PRT-100 Programmable Thermal Controller equipped with a hot bonnet without oil; each cycle consisted of 15 s at 95°C and 90 s at 70°C. Reaction products were pooled and immediately ethanol precipitated. The dried pellet was resuspended in 1.5 µl of formamide. The products were injected at 100 V cm−1 for 40 s. Short tandem repeat (STR) analysis Human blood samples were collected and DNA extracted. Polymerase chain reactions (PCR) were performed using 25 ng genomic DNA. Each PCR contained dNTPs (2 mM each dATP, dCTP, dGTP and dTTP), 5× N buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 170 µ ml−1 bovine serum albumin, 0.05% Tween 20, 0.05% NP-40 and 1.5 mMMgCl2) and 0.5 U Taq polymerase. STR primers were ordered from Research Genetics or Integrated DNA Technologies Inc., and were fluorescently labeled with 6-FAM, TET or HEX. The total PCR volume was 15 µl. The mixture was first subjected to an initial denaturing step of 94°C for 1 min. PCR was performed for 30 cycles consisting of 94°C for 1 min, 56°C for 2 min and 72°C for 1 min. The mixture was then treated to a final elongation step of 72°C for 7 min. PCR products were purified with Microcon-30 columns to remove excess salts and primers according to the manufacturers' instructions. The final elution was in formamide. TAMRA-labeled Genescan-500 sizing ladder was added to the purified PCR products and samples were denatured at 94°C for 2 min immediately prior to injection. The PCR products were separated by use of a denaturing 7% linear polyacrylamide solution in 1× TBE buffer with 6 M urea. The polymer was prepared as described above. The fused-silica capillaries were 43 cm long, 140 µm OD and 50 µm ID. Electrokinetic sample injection was 100 V cm−1 for 30 s. The sheath flow buffer was 1× 45°C. Subsequent to the run, data were analyzed using Igor Pro and MatLab. Figure 3 View largeDownload slide (A) Image of the GRIN lens array. The image was a back-illumination of the optical system. (B) Superimposed image of the fluorescence spots and back-illuminated spots seen through an auxiliary microscope placed opposite the sheath-flow cuvette from the collecting optic. Capillaries were 50 µm ID, 150 µm OD and 37.0 cm long, filled with 10 mM borate, pH 9.2. Fluorescein concentration was 10−7 M. The argon ion laser power was 4.0 mW. Green spots are fluorescence from sample streams, while the brown spots are scattering from the capillary tips. Figure 3 View largeDownload slide (A) Image of the GRIN lens array. The image was a back-illumination of the optical system. (B) Superimposed image of the fluorescence spots and back-illuminated spots seen through an auxiliary microscope placed opposite the sheath-flow cuvette from the collecting optic. Capillaries were 50 µm ID, 150 µm OD and 37.0 cm long, filled with 10 mM borate, pH 9.2. Fluorescein concentration was 10−7 M. The argon ion laser power was 4.0 mW. Green spots are fluorescence from sample streams, while the brown spots are scattering from the capillary tips. Data processing A simple base-calling algorithm was used to analyze the data. The routine was written in MatLab and run on a G3 power Macintosh and will be described in detail elsewhere. The routine has several components. The data were first convoluted through a Gaussian filter and then each of the traces was baseline corrected. Next, a response matrix was constructed based on the relative fluorescence intensities in each spectral channel. The data were multiplied by the inverse of this spectral response matrix to convert from spectral-space to dyespace (28). A mobility shift routine was incorporated to accommodate differences in mobilities for fragments labeled with the different dyes. Local maxima were identified and sequence was called based on the maximum dye response. Peak area was also calculated and used to identify and resolve multiplets late in the run. Results and Discussion Hydrodynamics In our sheath-flow cuvette, an array of capillaries is snugly fit into a rectangular quartz flow chamber. A simple siphon pumps the fluid through the interstitial spaces between the capillaries and draws the analyte streams, one per capillary, in the open region below the capillaries. A single laser beam excites fluorescence from all sample streams simultaneously. The highly transparent sheath fluid and the vanishingly low concentration of the DNA sample produces a negligible attenuation of the laser beam across the cuvette. Figure 3 presents a photograph of the sample streams. Each spot is ∼50 µm in diameter, which equals the inner diameter of the capillaries. The spots are uniformly spaced by ∼150 µm, which equals the outer diameter of the capillaries. The fluorescent spots are well separated in the photograph, generating negligible cross-talk. The design of a successful multiple-capillary sheath-flow cuvette requires careful attention to hydrodynamic focusing. In particular, it is necessary to have uniform sheath flow between each capillary. An unbalanced flow will cause the sample streams to deflect towards the region of lower flow velocity. This deflection of the sample stream results in misalignment with the optical system and can result in the failure to record a signal from that capillary. Uniform hydrodynamic flow is achieved if the capillaries are uniformly spaced within the cuvette.While it is possible to use micromachined cuvettes to hold the capillaries on uniform centers, our five-capillary instrument employs a somewhat simpler design that ensures uniform capillary spacing. The capillaries are inserted into a rectangular sheath flow cuvette. The narrow dimension of the cuvette is matched to the outer diameter of the capillary (Fig. 2). The inner walls of the cuvette are slightly tapered so that the top of the cuvette is slightly wider than the sum of the capillaries' diameters while the bottom of the cuvette is slightly narrower than that distance. As a result, the capillaries are squeezed together as they are inserted into the cuvette; since the capillaries are in contact, their spacing is very uniform, as are the sheath flow and the sample streams. Optics Microscope objectives are efficient collection optics for fluorescence detection in capillary electrophoresis (29). In our system, a 20 × 0.5 numerical aperture microscope objective, which provides a collection efficiency of 6.7%, collects fluorescence from the sheath flow cuvette. With 50-µm ID and 150-µm OD capillaries, the objective produces an image that consists of 1-mm diameter fluorescence spots at 3-mm spacing. A set of single-photon counting avalanche photodiodes is used for fluorescence detection. These very rugged devices provide extremely high quantum efficiency (>50%) and reasonable dynamic range. The APDs are housed in rather bulky containers, which contain low-noise amplifiers, Peltier coolers and single-photon detecting electronics. The APDs are too bulky to be used directly to detect the fluorescence images from the capillary array; we cannot pack them closely enough to simultaneously monitor fluorescence from each capillary. A set of five GRIN-lenses, coupled to fiber optics, is used to transmit fluorescence from the image-plane of the microscope objective to the APDs. The core of the optical fiber is only 100 µm in diameter, which is much smaller than the 1-mm fluorescence spot. We use a set of GRIN lenses, 1.8 mm diameter and 0.25 pitch, at the image plane at 3-mm center spacing to couple fluorescence into the optical fibers (Fig. 2). These inexpensive, compact optical elements efficiently couple fluorescence into the optical fibers. The use of optical fibers-GRIN lenses has proven to be valuable in alignment of the system. The optical fibers can be disconnected from the APDs. The detection end is illuminated with a lamp, creating back-illumination of the optical system. When viewed through an alignment microscope placed on the opposite side of the sheath flow cuvette from the collection optics, the illuminated optical fibers transmit light through the GRIN lenses to the 20× microscope objective into the sheath-flow cuvette (Fig. 3). Alignment is achieved by flowing dilute fluorescent dye through the capillaries; the relative position of the cuvette and the laser beam are adjusted until the fluorescent spots from the dye and the back-illuminated spots from the GRIN lenses are superimposed. The optical fibers are then reconnected to the APDs and a final tweaking of the optical system is performed to maximize the signal from the APDs. Detection limits The limit of detection was evaluated in free-zone-electrophoresis mode. The 37.0-cm capillaries were filled with a 10 mM borate buffer. Electrophoresis was performed with an electric field of 300 V cm−1 across the capillaries. A 1.1-nl plug of fluorescein was injected electrokinetically (1.0 kV, 5 s). Figure 4 presents an electropherogram of a 2 × 10−12M solution of fluorescein (2.2 zeptomol or 1300 molecules injected). The five traces were recorded simultaneously and presented as photon counts per 200 ms window. Migration of the fluorescein solution from the capillary generated one peak per capillary. The difference in migration time reflects the differences in electro-osmotic flow between the capillaries; the capillary walls were not coated for this experiment. The average peak area corresponds to 12 000 photons above the background signal level; each molecule generated an average of nine detected photons. Blank injections were performed by dipping the capillaries into the dye solution without application of injection potential; no peaks were observed from these blanks. Detection limits (3σ) were 130 ± 30 molecules (2 × 10−13 M) injected onto the capillaries (30). These detection limits reflect the good light collection efficiency of the optical system, the low background signal generated in the sheath-flow cuvette, and the high quantum efficiency and low-noise of the APDs. Figure 4 also demonstrates the other important feature of the design—the fluorescence detection is free of cross-talk; a peak in one capillary did not generate a signal in an adjacent capillary. The sheath flow not only lowered the background in fluorescence detection, but also provided excellent physical isolation for the separation channels even when the capillaries were in contact. Figure 4 View largeDownload slide Injection of 1300 fluorescein molecules. The capillaries were 50 µm ID, 150 µm OD and 37.0 cm long, filled with 10 mM borate, pH 9.2. Fluorescein, 2 × 10−12 M, was injected at 1 kV for 5 s. The electrophoresis was conducted at an electric field strength of 300 V cm−1. Argon ion laser power was 4.0 mW at 488 nm. Each data point was a 0.2 s count. The data were subjected to a binomial smoothing before plotting. Figure 4 View largeDownload slide Injection of 1300 fluorescein molecules. The capillaries were 50 µm ID, 150 µm OD and 37.0 cm long, filled with 10 mM borate, pH 9.2. Fluorescein, 2 × 10−12 M, was injected at 1 kV for 5 s. The electrophoresis was conducted at an electric field strength of 300 V cm−1. Argon ion laser power was 4.0 mW at 488 nm. Each data point was a 0.2 s count. The data were subjected to a binomial smoothing before plotting. DNA sequencing at 50°C By incorporating two-laser-line excitation and four-spectral-channel detection (9) into the five-capillary system, we turned the five-capillary instrument into a modest throughput, high-performance DNA sequencer. Figure 5 shows a typical sequencing separation performed at 50°C at a moderate electric field strength of 185 V cm−1 in a capillary filled with 6% non-cross-linked polydimethylacrylamide. The sequencing accuracy was 98.8% for 650 bases when using a simple base-calling algorithm written using MatLab. The software performance was limited by difficulties in handling multiplets late in the run; all errors were associated with an inaccurate estimate of these multiplets. Clearly, improved software will result in improved sequencing accuracy. Microsatellite analysis Markers on chromosome 7 were chosen to test the application of microsatellite methodology to capillary electrophoresis. The chromosome is ∼184 cM (sex-averaged) in genetic length; five microsatellite markers (D7S479, D7S500, D7S501, D7S523 and D7S554) were used to test the technology. These markers cover almost half of the long arm of the chromosome. Generally, markers spaced at ∼20 cM intervals allow the detection of linkage to a distance of 10 cM on either side of any putative disease-causing gene. Figure 6 presents five loci for a child and its parents, along with the signal from a commercial size standard. Although the D7S479 locus generated significant stutter bands, the patterns are clearly resolved and identification of the STR pattern is trivial. Figure 5 View largeDownload slide DNA sequencing run of an M13mp18 sample. The separation was performed in 6% non-crosslinked polydimethylacrylamide at 50 °C at an electric field strength of 185 V cm−1. The base-calls were performed using an algorithm written in MatLab. The called sequence is given above each peak. Errors are noted beneath the called sequence. Figure 5 View largeDownload slide DNA sequencing run of an M13mp18 sample. The separation was performed in 6% non-crosslinked polydimethylacrylamide at 50 °C at an electric field strength of 185 V cm−1. The base-calls were performed using an algorithm written in MatLab. The called sequence is given above each peak. Errors are noted beneath the called sequence. The microsatellite allele sizes for each family member determined by capillary electrophoresis were compared to those produced using traditional radioactive labeling and slab gel separation. The allele sizes generated by the two techniques corresponded (data not shown), indicating that DNA fragment sizes can be accurately determined and directly compared with the use of an internal standard. Separation of DNA fragments by capillary electrophoresis was rapid, with DNA of 500 bp length detected within 200 min with single base pair resolution. Conclusions We describe a five-capillary DNA sequencer based on a sheath-flow cuvette. The instrument uses avalanche photodiodes and a sheath-flow cuvette to produce extremely high detection sensitivity, which is important when analyzing small amounts of fluorescently labeled DNA. The instrument can operate at 50°C, which is valuable in reducing compressions in DNA sequencing. The instrument can also be used for genetic mapping, where the use of fluorescently labeled size markers facilitates comparison of genotyping patterns between individuals. The instrument currently relies on manual refilling of the capillaries with sequencing matrix between runs. Roughly 2.5 h were required to refill the capillaries and analyze the next sample. This turnaround time would be improved with an automated capillary refilling system, such as that found on commercial instruments. The instrument fills a niche between single-capillary DNA sequencers and 96-capillary DNA sequencers. We have also constructed 16- and 32-capillary versions of this instrument, which will be described elsewhere (H.J.Crabtree, S.J.Bay, D.Lewis, L.Coulson, G.Fitzpatrick, D.J.Harrison, S.Delinger, J.Z.Zhang, and N.J.Dovichi, paper submitted). Figure 6 View largeDownload slide STR analysis of five loci from a child and its parents. The blue peaks are size-markers that were used to align the patterns. The data were treated with a color-inversion matrix to correct for spectral overlap between the dyes. Data from the migration period containing each locus is plotted in the five panels. Figure 6 View largeDownload slide STR analysis of five loci from a child and its parents. The blue peaks are size-markers that were used to align the patterns. The data were treated with a color-inversion matrix to correct for spectral overlap between the dyes. Data from the migration period containing each locus is plotted in the five panels. Acknowledgements This work was supported by the Canadian Genetic Diseases Network, the Canadian Bacterial Diseases Network, the Natural Sciences and Engineering Research Council of Canada (NSERC) and Sciex. K.V. acknowledges a graduate fellowship from the Alberta Heritage Foundation for Medical Research. 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TI - A multiple-capillary electrophoresis system for smallscale DNA sequencing and analysis
JF - Nucleic Acids Research
DO - 10.1093/nar/27.24.e36
DA - 1999-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-multiple-capillary-electrophoresis-system-for-smallscale-dna-wxtYRelcrM
SP - e36
EP - e42
VL - 27
IS - 24
DP - DeepDyve
ER -