Ultrafast STR Analysis by Microchip Gel Electrophoresis
Dieter Schmalzing, Lance Koutny, Aram Adourian, Paul Matsudaira, and Daniel
Ehrlich*
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142
Phillip Belgrader
Armed Forces DNA Identification Laboratory, Armed Forces, Institute of Pathology,
Rockville, MD 20805
* corresponding author
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ABSTRACT
Allelic profiling of the quadruplex short tandem repeat (STR) system CTTv has been routinely performed in less than two minutes on a microchip gel device with high accuracy. This represents an increase in speed of ten- to one hundred-fold when compared to capillary or slab gel electrophoresis, respectively. The extremely narrow injection plugs and short separation distances possible in microchips allow for such high separation speeds. The separations were performed in a replaceable polyacrylamide matrix under denaturing conditions at 50°C. Off-line robotics were used for fully automated sample preparation. Based on these results it can be envisioned that a single microchip array system will analyze hundreds of blood samples per hour in an entirely automated format.
INTRODUCTION
The analyses of short tandem repeats (STRs) is usually performed by conventional slab gel electrophoresis. While parallel, throughput is limited by the slow speed of the electrophoretic process which is in the range of two to three hours (1,2). The process is also labor intensive.
Capillary gel electrophoresis is currently being explored by a number of groups as a possible alternative to the conventional slab gel format (3). The use of capillary tubes with small internal diameters (< 100
mm) allows for high field strengths leading to fast separations. Moreover, the entire capillary based analysis process can be fully automated through the use of replaceable sieving matrices, on-line laser induced fluorescence detection and automatable sample injection. Separation times of ten to thirty minutes have been achieved on such capillaries for the analyses of single loci (4-7). Despite a nearly ten-fold increase in analysis speed, no increase in throughput is realized, since only one sample at a time can be analyzed. To improve STR throughput, bundles of capillaries were used (8,9). Despite the relative improvement in speed it can be speculated that a capillary-based approach is inherently constrained in speed and resolution. First, there seems to be a practical limit to which the length of the separation columns can be scaled down because the manipulation of short columns (less than 3 cm) becomes rather challenging, especially when employed in capillary arrays. Second, in many cases the length of the injection plug might be larger than that required for optimal performance due to the electrokinetic injection method used in gel filled capillaries. Third, the handling of a large number of columns may pose operational difficulties and might also limit the compactness of the device.In 1992 microchip based electrophoresis was introduced by Manz and Harrison (10,11). In this method, electrophoretic separations are performed in microchannels layed out inside of glass, fused silica or plastic substrates. Analysis times in the order of seconds have been reported on such devices for various DNA preparations (12-15). Those fast separations become possible for two reasons. First, since the sample and buffer reservoirs are fully integrated, very short separation distances can be used. Second, the total control over channel and intersection geometries permits the use of unique injection methods with extremely narrow injection plugs. In addition, the production and operation of dense arrays of microchannels for high throughput applications should be straightforward.
Our group has recently reported a systematic study of the performance of a microchip device used for the analysis of the quadruplex STR system CTTv (consisting of the loci vWA, TH01, TPOX, CSF1PO) (16). The influence of parameters such as temperature, field strength, diffusion, channel length and injector size on chip performance were investigated. In this paper we focus on practical aspects of our device.
MATERIALS AND METHODS
Micromachining. Electrophoretic microchip devices were fabricated as described in (16) utilizing standard photolithographic and chemical etching methods to produce channel structures in fused silica wafers.
Coating. The surfaces of the etched channels were chemically modified by covalent attachment of linear
polyacrylamide as reported (16) to stop electroosmotic flow and to prevent non-specific interactions.
Separation Matrix. The working buffer consisted of 1 X TBE with 3.5 M urea and 30% v/ v formamide (Pharmacia, Uppsala, Sweden). The separation matrix was 4% (w/ v) non-crosslinked linear polyacrylamide synthesized in-house by redox polymerization (16). The matrix was stored at 4
°C and loaded with a syringe into the coated microchip channels.Robotic Sample Preparation. The sample preparation was fully automated (17). In brief, bloodstain card punches in 96-well Cycleplates (Robbins Scientific, Sunnyvale, CA) were washed with FTA Purification Reagent (Fitzco, Mineapolis, MN), TE (1 mM Tris-HCL, 0.5 mM EDTA, pH 8.0), and ethanol. A volume of 30 mL of PCR mix 1x STR buffer (Promega, Madison, WI), 5’-fluorescein labeled CTTv Quadriplex primer pairs (5 mM of each primer) (Promega), bovine serum albumin (60 mg/mL) and AmpliTaq Gold® DNA polymerase (50 mg/mL) (Applied Biosystems Division-Perkin Elmer, Foster City, CA) was added to each well, followed by 25 mL of liquid wax (MJ Research, Watertown, MA). Thermal cycling was attained at 95
°C for 10 min, 10 cycles of 94°C for 1 min, 60°C for 1 min, and 70°C for 1.5 min, 20 cycles of 90°C for 1 min, 60°C for 1 min, and 70°C for 1 min, and 70°C for 10 min.Instrumentation. A schematic of the microchip genotyping apparatus is shown in Fig.1. A fused silica microchip was mounted on a temperature controlled stage with high voltage connections and optical access for laser induced fluorescence (LIF) detection. The microchip was affixed to a an Al2O3 alumina heater block whose temperature was controlled by an Omega Instruments (Stamford, CT) temperature controller and a series of thermocouples. The alumina block contained a machined aperture to allow for optical access of the detection zone.
High voltage was provided to platinum wire electrodes mounted in the four glass fluid reservoirs by a Spellman SL150 power supply (Plainview, NY). The voltages applied to each reservoir were controlled by a manual switching circuit and resistor - based voltage divider network. Actual applied voltages were monitored with a Keithley Instruments 614 electrometer with a high voltage probe. Laser - induced fluorescence detection was achieved using an argon ion laser operating with 4 mW of output power at 488 nm. The beam was focused to a spot size of 15 um in the channel at 30 degree angle of incidence with a 10 cm focal length lens. A 50X 0.45 N.A. long working distance microscope objective (Bausch & Lomb) collected the fluorescence emission. The collected light was spatially filtered by a 4 mm diameter aperture in the image plane and optically filtered by two 520 nm bandpass filters (520DF20 Omega Optical, Brattleboro, VT) and detected by a photomultiplier detection system. The PMT current signal was converted to voltage across a 10 kohm resistor, digitized with a PC-controlled 20 bit data acquisition system (Data Translation model 2802, Marlborough, MA) and analyzed using C Grams software (Galactic Industries, Salem, NH).
Microchip Gel Electrophoresis. The chip, freshly filled with the 4% polyacrylamide solution, was pre-electrophoresed for 3 min across the separation channel at 200 V/ cm and 50
°C. Separations were carried out under identical conditions. The chip was operated in the pinched cross injection mode producing an injection plug length of 100 mm and an injection volume of 0.3 nL.Four
mL of PCR amplified sample were added to 2 mL CTTv ladder and diluted to a total volume of 10 mL with 2X TBE buffer containg 3.5 M urea and 30% v/ v formamide. The samples were briefly vortexed, denatured for 2 min at 95°C, chilled on ice and pipetted into the microchip sample reservoir.Capillary Column Gel Electrophoresis. The fused silica capillary (i.d. 75
mm) was 50 cm long with a separation length of 37 cm. The column was coated by the same procedure as the microchip. The same matrix and the same electrophoretic conditions as in the chip device were used. Electrokinetic injection was performed from water samples at 200 V/ cm for 10 seconds.Slab Gel Electrophoresis. Three microliters of either 5’-fluorescein labeled CTTv ladder (diluted 1:5) was mixed with 3 mL of formamide containing 5’-Rox labeled Genescan-2500 size standard (Applied Biosystems Division Perkin Elmer). The sample was heated at 95 C for 2 min, quickly cooled in ice, and electrophoresed for 2.5 hours at 28 watts through a denaturing 8% polyacrylamide gel in Applied Biosystems 373 DNA sequencer running Genescan software. The sizes of the CTTv ladder and PCR products were automatically determined by Genescan analysis software using the local Southern method.
RESULTS
A schematic of the chip used for fast genotyping, is depicted in Figure 2. The device had a simple cross-structure. The three channels originating from the sample, the buffer and the sample waste reservoirs were 5 mm long. The separation channel had a length of 30 mm from the intersection to the waste reservoir. The detector was placed 26 mm from the injection point. The chip was wet-etched to a depth of 45mm, producing a half cylindrical channel with a width of 100mm at the top due to the isotropic etching conditions and the original 10mm pattern width. The cross sectional area of the channels was equivalent to that of a cylindrical capillary with a diameter of 70mm. The channel junction geometry produced an injection plug of only 100mm in length. The injections were performed in the pinched mode, schematicaly described in Figure 3. In addition to allowing for a small and reproducible injection volume, the pinched injector prevents diffusion of sample into the separation channel during operation.
Figure 4a shows the separation of the standard CTTv ladder performed on our microchip apparatus at 200 V/ cm in a 4% polyacylamide matrix under denaturing conditions at 50
°C. The ladder spans the range from 140 to 330 bases. The alleles in any given locus differ in length by 4 base pairs. On our device the separation is performed in less than two minutes with good resolution. The resolution ranges from 1.7 for the first set of alleles (vWA locus) to 1.1 for the last set (CSF1PO locus). For genotyping, the CTTv ladder was individually spiked with PCR amplified samples of several individuals. In all the cases, the alleles could be assigned without any ambiguity in under two minutes and the results were in perfect agreement with slab gel electrophoresis data. A typical result for one of the individuals is shown in Figure 4b. The person is homozygous for vWA (allele 14) and heterozygous for TH01 (alleles 7/ 9), TPOX (alleles 8/ 9) and CSF1PO (alleles 10/ 14).To illustrate the superior performance of our microchip gel device over slab or capillary gel electrophoresis, the analyses of one of the PCR amplified samples by these 3 electrophoretic formats are compared in Figure 5. The separations show similar resolutions, but the slab gel system needed approximately two hours to complete the separation whereas the capillary system used only approximately 1/4 of the time to produce the same result. This improved speed of analysis can be attributed to the high field strengths applicable in capillary devices. However, when compared to the microchip analysis, both systems appear excessively slow. The microchip analysis is a factor of 10 or 100 times faster than the capillary or slab gel analysis, respectively. The microchip system combines the high field strength of the capillary format with the advantage of ultra short injection plugs which in turn lead to ultra short separation distances.
CONCLUSION
We have shown that the STR analyses of the four loci STR system CTTv can be performed reliably in under two minutes in a single lane microchip device. Very high throughput should be obtainable with a dense array of such microchannels, which, when integrated with robotics for sample preparation, sample loading and gel replacement, should lead to a fully automated device for genotyping.
ACKNOWLEDGMENT
We thank Jonathan K. Smith for technical assistance and Lt. Col. Victor W. Weedn for helpful discussions. This work was supported by the National Institute of Health, the Defense Advanced Research Projects Agency, and the Air Force Office of Scientific Research. The views stated here are the opinions of the authors and in no way reflect the position of the U.S Army, the U.S. Air Force, or the Department of Defense.
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Figure 1. Schematic of the of microchip genotyping apparatus with laser - induced fluorescence detection optics and temperature - controlled heating block.
Figure 2. Schematic of the microchip architecture used for genotyping
Figure 3. Illustration of the pinched injection
Figure 4. a) Microchip electropherogram for the four - loci CTTv allelic sizing standard. b) Microchip electropherogram presenting the allelic profile of an individual obtained by spiking a PCR amplified sample with the CTTv sizing standard. Allele numbers are given above the peaks. Electrophoretic conditions as described in the text.
Figure 5. Comparison of nearly optimized methods for electrophoretic analysis of the same CTTv sample. Shown are results near optimal conditions for: (a) a gel plate (ABI 373, 28 Watts, 8% PAA gel, Genescan software), (b) a capillary (38-cm effective length, 200 V/cm, 4% PAA, 50
°C) and (c) a microchip (26-mm channel length, 100-mm injector length, 200 V/cm, 50°C, 4% PAA). The microchip result shows a fine structure due to the CTTv ladder that is added as an internal standard. As determined in the present study, there remains some opportunity to further optimize the microchip through reduction of the injector length. Note the large differences in run time for comparable quality in the separations.
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