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Clinical Chemistry 45: 1906-1917, 1999;
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(Clinical Chemistry. 1999;45:1906-1917.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Molecular Diagnostics on Microfabricated Electrophoretic Devices: From Slab Gel- to Capillary- to Microchip-based Assays for T- and B-Cell Lymphoproliferative Disorders

Nicole J. Munro1, Karen Snow2, Jeffrey A. Kant3 and James P. Landers1,4,a

1 Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260.

2 Division of Laboratory Genetics, Mayo Clinic, Rochester, MN 55905.

3 Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213.

4 University of Pittsburgh Cancer Institute, Pittsburgh, PA 15260.
a Address correspondence to this author at: Department of Chemistry, University of Virginia, Charlottesville, VA 22901. Fax 804-243-8852; e-mail jpl5e{at}virginia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Current methods for molecular-based diagnosis of disease rely heavily on modern molecular biology techniques for interrogating the genome for aberrant DNA sequences. These techniques typically include amplification of the target DNA sequences followed by separation of the amplified fragments by slab gel electrophoresis. As a result of the labor-intensive, time-consuming nature of slab gel electrophoresis, alternative electrophoretic formats have been developed in the form of capillary electrophoresis and, more recently, multichannel microchip electrophoresis.

Methods: Capillary electrophoresis was explored as an alternative to slab gel electrophoresis for the analysis of PCR-amplified products indicative of T- and B-cell malignancies as a means of defining the elements for silica microchip-based diagnosis. Capillary-based separations were replicated on electrophoretic microchips.

Results: The microchip-based electrophoretic separation effectively resolved PCR-amplified fragments from the variable region of the T-cell receptor-{gamma} gene (150–250 bp range) and the immunoglobulin heavy chain gene (80–140 bp range), yielding diagnostically relevant information regarding the presence of clonal DNA populations. Although hydroxyethylcellulose provided adequate separation power, the need for a coated microchannel for effective resolution necessitated additional preparative steps. In addition, preliminary data are shown indicating that polyvinylpyrrolidone may provide an adequate matrix without the need for microchannel coating.

Conclusions: Separation of B- and T-cell gene rearrangement PCR products on microchips provides diagnostic information in dramatically reduced time (160 s vs 2.5 h) with no loss of diagnostic capacity when compared with current methodologies. As illustrated, this technology and methodology holds great potential for extrapolation to the abundance of similar molecular biology-based techniques.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DNA fragment analysis has become an integral component of clinical diagnostics. Detection of PCR-amplified fragments currently is used for a wide variety of clinical applications such as disease diagnosis and minimal residual disease detection (1)(2). Separation of DNA fragments has traditionally been performed by electrophoresis on slab gels utilizing densitometry or Southern blot detection. The potential for translating these separations to capillary electrophoresis (CE)1 has been illustrated (3). In addition to the minute quantities of sample and reagents required for CE, there is a dramatic reduction in analysis time. This time reduction results from the translation of the electrophoretic format from the slab gel to the capillary, where higher applied fields can be tolerated as a result of the rapid heat transfer through the micron-scale internal diameter capillary. Unfortunately, incorporating parallel processing, as is performed on slab gels, has not been as prolific, although several examples have been shown [for example, see Ref. (4)] and commercial instruments are available. For high-throughput analysis applications such as genomic sequencing, clinical diagnostics, or drug screening, parallel processing will be essential.

A miniaturized variant of CE exploits extant microfabricated chip technology to create an electrophoretic chip device that provides even greater advantages for rapid and cost-effective clinical analysis. Perhaps the greatest advantage of the electrophoretic chip platform over the slab gel and even the capillary formats is the potential for integrating sample processing steps directly (5)(6)(7)(8). In combination with parallel processing, this has the potential to create the rapid, high-throughput platform demanded by current clinical diagnostic applications. The potential impact on the clinical sector for electrophoresis using microfabricated devices has been discussed (9) with supporting preliminary data from applications such as serum protein separations (10) and immunoassays (11)(12)(13). Clinically relevant DNA separations on microfabricated devices have also been shown, including the separation of PCR products of the dystrophin gene (14) and DNA-sequencing products (15).

The development of electrophoretic microchip technology is in its infancy, although an increased number of research groups are reporting their production and use (5)(6)(8)(11)(12)(13)(14)(15)(16)(17)(18)(19). Despite the fact that few reports have demonstrated the application of electrophoretic microchips to real-world analysis, it is clear that this technology is ideally suited to the rapid analysis of PCR-amplified DNA.

A multitude of diagnostic assays currently in use incorporate PCR-based amplification for detection of aberrant DNA sequences (mutations, rearrangements, deletions, and translocations) correlative with the onset of disease (1). Such is the case for the diagnosis of T- or B-cell lymphoma, where PCR results can be available within 1 day whereas Southern blot studies require 1 week (20). Clinical diagnosis of T- or B-cell lymphoproliferative disorders takes advantage of the maturation process of the T and B cells by focusing on the analysis of the genes encoding for the T-cell receptor (TCR) and immunoglobulin heavy chain (IgH). The majority of oncogenic events begin after rearrangement (21); therefore, the offspring of a transformed cell will possess the same specific gene sequence produced when the parent cell underwent rearrangement. A predominance of a single sequence will, therefore, be a mark of clonality, an indication of malignancy. However, as a result of the combinatorial nature of the rearrangement process, a normal cell population will possess a polyclonal population, a diverse variety of low abundance sequences. PCR-based assays have been developed to analyze the lengths of the DNA fragments produced when a variety of primers for the variable and joining regions are used (multiplexed PCR) (22). When multiplexed PCR and subsequent fragment separation are used, a normal cell population displays a large variety of DNA fragment sizes, whereas a malignant population displays a predominance of one DNA fragment size.

As with other PCR assays, established protocols for analyzing B- and T-cell amplified products have relied on slab gel electrophoresis to identify products and provide semiquantitative data. CE provides a more rapid, automated platform for high-resolution PCR fragment analysis with excellent detection sensitivity provided by laser-induced fluorescence (LIF) detection (23). In an earlier report, Oda et al. (24) took the first steps toward utilizing CE for T-cell malignancy diagnosis by defining initial conditions for separation of the PCR-amplified fragments. In this report, this work is extended to include the analysis of B-cell malignancy assay samples and the methodology translated to the microfabricated chip, where separations can be achieved over an order of magnitude faster. Comparisons are made between slab gel, capillary, and microchip electrophoretic results with respect to analysis time, resolution, and diagnostic capabilities. In addition, with a vision toward a simple and robust microchip platform for diagnostic DNA fragment detection, the results of separations that were performed in a microchip containing uncoated channels are presented; these results, although preliminary in nature, illustrate the feasibility for this methodology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
sieving matrix preparation
A 1x Tris-borate-EDTA [TBE; 89 mmol/L Tris (Sigma Chemicals), 89 mmol/L boric acid (EM Chemicals), and 2 mmol/L EDTA (Sigma)] solution was adjusted to pH 8.6 with 2 mol/L sodium hydroxide. Hydroxyethylcellulose (HEC, Mr 250 000; Aldrich) and polyvinylpyrrolidone (PVP, Mr 360 000; Acros) sieving matrixes were prepared by heating a 1x TBE solution to the point were condensation was seen on the inside glass surface (~55 °C) and then adding the polymer (10 or 20 g/L) slowly to the rapidly stirring solution. Heated mixing at ~55 °C continued until the solution cleared (~15 min); it was then allowed to stir for 1 h with no heating. The solution was filtered through an 0.8 µm filter (Millipore) and stored at 4 °C. Before the sieving matrix was used, 1-(4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-quino-linium)-3-trimethyl-ammonium propane diiodide (YO-PRO-1; Molecular Probes) was added at 1 µL of YO-PRO per milliliter of sieving matrix (a 1:1000 dilution of the 1 mmol/L solution obtained from the manufacturer).

instrumentation
CE instrumentation.
A Beckman P/ACE System 5510 (Beckman Instruments) was used for CE analysis. Fluorescence detection used a P/ACE System Laser Module 488 with a P/ACE LIF detector, which excites at 488 nm and collects emission at 520 nm ± 10 nm. A laser power of 0.25 mW was presented at the capillary. Instrument control and data collection were performed with an IBM 486 ValuePoint computer utilizing System Gold software (Ver. 8.1).

Electrophoretic microchip instrumentation.
The separation voltages were supplied by an in-house manufactured high-voltage power supply controlled by a program written in Labview. For fluorescence detection, the 488-nm line of an argon ion laser (532R-BS-AO4; Melles Griot) was expanded to fill the back aperture of an objective. The expanded beam was reflected off a beam splitter (505DRLP02; Omega Optical) set at 45° to the incident beam and into the channel of an electrophoretic microchip by an objective (20x/numerical aperture, 0.5). Fluorescence emitted by the sample was collected by the objective and focused by a 200-mm lens onto a 400-µm pinhole (25). The fluorescence wavelength was spectrally filtered by a 530 nm bandpass filter (Omega Optical 530DF30, FWHM 30 nm), collected by a photomultiplier tube (PMT, Hamamatsu R38960), and processed via a program written in Labview.

Electrophoretic microchip microfabrication.
Electrophoretic chips were microfabricated by the Alberta Microelectronics Center. Channels were etched in one glass plate to which a glass top plate was bonded. Electrophoretic chips consisted of an eight-channel arrangement, with each channel composed of an injection cross with a sample-to-sample waste distance of 1.45 cm, inlet-to-outlet distance of 6.65 cm, and the junction 0.5 cm from the inlet (Fig. 3 ). Detection occurred 4.2 cm from the injection cross. The sample channel was 100 µm wide and 10 µm deep, and the separation channel was 50 µm wide and 10 µm deep. One-millimeter holes were drilled in the top glass plate to allow access to the channels. Cut pipette tips were epoxied onto the top plate to form larger wells for solutions and placement of gold-coated electrodes.



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  		Figure 3. Configuration of the electrophoretic microchip (A) and layout of the confocal fluorescence system for on-chip detection (B).

PMT, photomultiplier tube.

pcr amplification of tcrg rearrangements
Rearranged T-cell receptor-{gamma} (TCRG) gene sequences were amplified using multiplexed PCR as described previously (20). For the TCRG variable segments, five primers (TCRG V2, 3, 4, 8, and 9) were used along with three primers for the TCRG joining segments (JGT12 with consensus sequences for J1.3 and J2.3, JGT3 with consensus sequences for J1.1 and J1.2, and JGT4 with consensus sequences for J1.2). The PCR reaction mixture included 0.75 U of Taq polymerase (Taq Gold-P; Perkin-Elmer), 200 µmol/L each of dATP, dCTP, dGTP, and dTTP, 1.5 mmol/L MgCl2, and 1.0 µmol/L of each primer in standard PCR buffer (Perkin-Elmer) for a total volume of 25 µL. PCR cycling conditions included a 10-min hold at 95 °C, followed by 40 cycles of PCR (94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min) and a 10-min extension at 72 °C.

pcr amplification of igh rearrangements
IGH PCR was performed in 25-µL reactions using 1 U of Taq polymerase (PE Biosystems) and a consensus 5' framework III variable region primer (AGG TGC AGC TGG TGC AGT CTG G) with a mixture of three consensus primers directed at heavy chain joining regions 1, 2, 4, and 5 (ACC TGA GGA GAC GGT GAC CAG GGT), 3 (TAC CTG AAG AGA CGG TGA CCA TTG T), and 6 (ACC TGA GGA GAC GGT GAC CGT GGT). PCR thermocycling conditions included an initial denaturation for 5 min at 95 °C, followed by 35 cycles of PCR (95 °C for 15 s, 52 °C for 20 s, 72 °C for 30 s) and a 5-min extension at 72 °C.

analysis via slab gel electrophoresis with sybr green i staining
For the TCRG gene rearrangement assay, 3.5 µL of the amplified PCR mixtures was loaded onto an 8% polyacrylamide (19:1, by weight) minigel. The DNA marker was pGEM (Promega). Electrophoresis was performed using a 1x TBE buffer for 75 min at 200 V. Gels were stained with SYBR Green 1 (1 µg/L; Molecular Probes) with gentle agitation for 30 min at room temperature. Stained gels were illuminated with a 300 nm ultraviolet transilluminator and photographed with Polaroid 667 black and white print film through a SYBR Green I gel stain photographic filter (1- to 2-s exposure; f-stop, 4.5).

For IGH gene rearrangement PCR assays, 30 µL of PCR reaction product mixed with 3 µL of blue sucrose was loaded into lanes of an 8% polyacrylamide gel (16 cm x 20 cm x 1.5 mm) and electrophoresed in 1x TBE buffer for 2.5 h at 180 V. The gel was stained with SYBR Green I and photographed with type 57 Kodak film. MspI digests of pBR322 (New England Biolabs) were used as molecular weight markers.

pcr product analysis via lif-ce
For CE separations using HEC (described above), a 37 cm x 50 µm (effective length, 30 cm) fluorocarbon (FC)-coated µ-SIL capillary (J & W Scientific) was used. YO-PRO-1 (1 µmol/L) was used as a fluorescent intercalator. The DNA marker, HaeIII digest of pBR322 (Boehringer Mannheim Biochemicals), was diluted 1:100 with 10 mmol/L Tris, 1 mmol/L EDTA for a final concentration of 4.20 mg/L. The PCR-amplified samples were diluted 1:10 in 10 mmol/L Tris, 1 mmol/L EDTA. FC-coated capillaries were first rinsed with 20 column volumes of water. HEC separations used the following method: 3-s electrokinetic injection of water at 3.5 kV, 10-s injection of sample at 8.5 kV (230 V/cm), 3-s injection of water at 3.5 kV. Separation followed at 8 kV (216 V/cm), using reversed polarity (inlet cathode, outlet anode) and maintaining the capillary at 20 °C. The capillary was rinsed with 5 column volumes of water between runs and prerinsed with 10 column volumes of fresh (unelectrophoresed) buffer before each run.

For CE separations using PVP (described above), 37 cm x 50 µm bare silica capillaries (Polymicro Technology) were first conditioned by rinsing with 20 column volumes of water. Injection and run settings were as stated for the HEC/FC-capillary system.

pcr product analysis via microchip electrophoresis
Microchip channels were coated with linear polyacrylamide (PA) by a modified Hjertén method (26). Samples were desalted by placing 10 µL of undiluted sample in a Microcon® YM-10 Centrifugal Filter Device (Amicon) with 90 µL of water and then centrifuged at 7g for 10 min. One hundred microliters of water was then added, vortex-mixed, and centrifuged for 10 min at 7g; this procedure was repeated five times, and the eluent was discarded. To reconstitute the sample, 10 µL of water was added, vortex-mixed, centrifuged for 30 s at 2g; the cartridge was then inverted and centrifuged at 7g for 2 min. The same HEC and PVP sieving matrices were used as described for the CE analyses. Channel preparation and between-run rinses were performed as described for the capillary system. Microchip sample injection was performed by applying a 400 V (275 V/cm) potential across the sample and sample waste reservoirs, with the sample at ground. For separation, the sample and sample waste were grounded and -200 V was applied to the inlet and 900 V to the outlet (165 V/cm). Fluorescence was collected at 10 Hz.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
slab gel and ce analysis of tcrg and igh gene rearrangements
Conventional methodology for detecting gene rearrangements consistent with T- and B-cell lymphomas involves electrophoresis of T-cell receptor-{gamma} (TCRG) and immunoglobulin heavy chain (IGH) gene rearrangement PCR products on an acrylamide gel. Fig. 1 shows SYBR Green-stained polyacrylamide gels for these assays. In these analyses, a normal polyclonal population of lymphoid cells will yield a series of DNA fragments that appear as a "smear" or a series of discrete bands, depending on the resolution. Conversely, a clonal lymphocyte population, indicative of a lymphoid neoplasm, will yield one or more dominant bands. The PCR primers used for multiplexed amplification of the TCRG variable region(s) yield fragments in the 150- to 250-bp range (Fig. 1A ). The polyclonal fragments typical of a normal sample (negative for a T-cell lymphoproliferative disorder) are shown in lane 9 (negative control) and in lane 6 (patient sample). Both cases display the smear of DNA fragments typical of polyclonal populations. In contrast, the presence of a dominating DNA fragment indicative of clonality is illustrated by the patient samples in lanes 2, 3, and 4, which exhibit one or more densely stained bands in the 150- to 250-bp region and compare favorably with the positive controls in lanes 7 and 8. Samples in lanes 1 and 5 were negative/equivocal for clonality.



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  		Figure 1. Slab gel separations of TCRG and IGH gene rearrangement products.

(A), slab gel of nine TCRG gene rearrangement products and a DNA ladder, pGEM. Lanes 1 and 5 were diagnosed as negative equivocal; lanes 2–4, 7, and 8 as positive; and lanes 6 and 9 as negative. Lane 7 shows the results for a positive control that contains a 5% dilution of DNA from a clonal population into genomic DNA from white blood cells. Lane 8 represents a positive control containing a 10% dilution of DNA from the same clonal population into DNA from thymus. Lane 9 is a negative control. (B), slab gel of IGH gene rearrangement products and a DNA ladder, pBR322/Msp1. Lanes 1, 5, 7, and 8 were diagnosed as positive for monoclonality; lanes 2–4 and 6 as negative for monoclonality. Lanes 1 and 2 are controls.

Interrogation of B-cell populations via the same molecular approach is shown in Fig. 1BUp . Here, multiplex PCR amplification of the variable region(s) of the IGH gene yields fragments in the 80- to 140-bp region (Fig. 1BUp ). As with the T-cells, normal samples produce a polyclonal population of DNA fragments that are observed as low-abundance DNA bands spanning the 80- to 140-bp range (lanes 2–4 and 6). The lower molecular weight fragments yielded by the IGH gene amplification are resolved better than the corresponding T-cell amplification products and, therefore, are not observed as a smear but rather as a series of discrete bands. However, with B-cell samples that display clonality, dominant band(s), which signal the presence of the monoclonal population, are clearly observed and indicative of a B-cell lymphoproliferative disorder (lanes 1, 5, 7, and 8).

The results from CE analysis of selected samples analyzed via slab gel electrophoresis in Fig. 1Up are shown in Fig. 2 . CE conditions included a 37 cm x 50 µm FC-coated capillary containing 10 g/L HEC solubilized in 1x TBE buffer at pH 8.6. A fluorescent intercalator, YO-PRO-1, was added to the buffer for LIF detection. Fig. 2A shows CE separations representative of TCRG gene rearrangement products and compares these electropherograms with a separation of the lower molecular weight fragments in a pBR322 HaeIII digest (bottom panel). The top panel (T1) shows a DNA fragment pattern indicative of a polyclonal population, with the stained acrylamide gel (inset) displaying an equivalent banding pattern. Because of the band (gel) and peak (capillary) indicated by the arrows, this patient was diagnosed as negative/equivocal. A monoclonal fragment pattern is illustrated in the middle panel (T2, with acrylamide gel inset). For this sample, the diagnosis is obvious, based on the presence of a dominant band on the gel and the corroborative peak in the electropherogram. The corresponding PCR-amplified IGH rearrangement products were also analyzed by CE, and the results are shown in Fig. 2B along with the pBR322 HaeIII digest marker (bottom panel). The top panel (B1) shows a positive sample, with a broad band on the gel being partially resolved into two peaks by CE; this is unequivocal identification of a monoclonal population. Conversely, the middle panel (B2) illustrates a characteristic profile for a sample negative for a B-cell malignancy, with the widespread polyclonal population of fragments displayed as a sawtooth pattern produced by the high-resolution separation.



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  		Figure 2. CE separation of gene rearrangement products.

(A), negative/equivocal sample (T1) and positive (T2) TCRG gene rearrangement samples analyzed via CE with corresponding slab gel separations (insets) and DNA marker, HaeIII digest of pBR322. (B), CE separation of a positive (B1) and negative (B2) IGH gene rearrangement sample with analogous slab gel separations (inset) and DNA marker. Electropherogram labels correspond to those in Fig. 1Up . Conditions: 37 cm x 50 µm FC capillary, effective length, 30 cm; 10 g/L HEC in 1x TBE; 10 s electrokinetic injection at 8.5 kV; separation at 8 kV.

microchip-based electrophoretic analysis of tcrg and igh gene rearrangements
Efficient microchip-based electrophoretic analysis requires a multichannel electrophoretic microchip and an optical apparatus capable of sensitive fluorescence detection (Fig. 3Up ). The microchip illustrated in Fig. 3AUp contains eight independent microchannels with the corresponding reservoirs for each. Although parallel analysis can be performed, only single-channel analysis was required in this initial study. Injection of DNA was accomplished electrokinetically at the intersection between the separation channel (50 µm wide and 10 µm deep) connecting the inlet and outlet reservoirs and the cross channel (100 µm wide and 10 µm deep) that connects the sample and sample waste reservoirs. The sample reservoir-to-sample waste distance was minimized to negate any electrophoretic artifacts that may result from separation in the cross channel itself during injection. With a separation distance (cross channel intersection-to-detection point) of 4.2 cm, the effective length of the separation channel was sevenfold shorter than the capillary used to generate the separations shown in Fig. 2Up . Detection was accomplished by focusing the laser beam on the center of the microchannel at the detection window of the microchip (shown in Fig. 3AUp ). Fig. 3BUp shows the configuration of the home-built confocal epifluorescence microscopic detection system. The system uses an argon ion laser that is filtered spectrally and expanded to fill the back aperture of a microscope objective lens to focus the beam tightly inside the channel. Fluorescence is collected via the same objective lens and, after passage through the optical components displayed in Fig. 3BUp , is detected by a photomultiplier tube. A single software program controls both the detection system and the electrophoresis high-voltage system, synchronizing the separation with the detection. This configuration is ideal for microchip-based electrophoresis because it allows for sensitive detection while leaving the microchip surface completely accessible for manipulation of sample, solutions, and electrophoresis hardware.

Microchip-based electrophoretic separation of DNA markers.
After the microchannels were coated with PA, the microchannels were filled and equilibrated with 10 g/L HEC in 1x TBE containing micromolar concentrations of YO-PRO-1 as an intercalating dye for double-stranded DNA detection. A typical separation of DNA markers, HaeIII digest of pBR322, produced the microchip electropherogram shown in Fig. 4 A. The high resolution observed is comparable to separation of the same DNA markers in the 37 cm x 50 µm capillary (Fig. 4B ).



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  		Figure 4. Separation of the HaeIII digest of pBR322 using microchip (A) and CE (B) with 10 g/L HEC in 1x TBE and an FC-coated capillary and PA-coated channel.

Other conditions as stated in text.

Analysis of TCRG and IGH gene rearrangements.
The samples evaluated by gel electrophoresis (results shown in Fig. 1Up ) were also analyzed by microchip electrophoresis with optimized conditions for sample preparation (sample desalting and a 1:10 dilution in 10 mmol/L Tris, 1 mmol/L EDTA). Fig. 5 shows a comparison of the capillary and electrophoretic microchip formats for separation of the TCRG gene rearrangement products for four samples. The profile for sample T1 (Fig. 5B ) displays the same series of low-abundance fragments (fragment size range, 150–250 bp) as observed for gel and capillary separations and also allows for detection of the suspicious single band (fragment size, ~140 bp on the gel; indicated by the arrow in Fig. 5 ) that led this sample to be classified as negative/equivocal. A typical negative sample profile is displayed for sample T4, which contrasts the profiles for positive samples T2 and T3 that clearly demonstrate clonality.



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  		Figure 5. Capillary and microchip electrophoresis of TCRG gene rearrangement products.

Capillary (A) and microchip (B) separations of samples labeled in Fig. 1AUp . Sample T1 was diagnosed as negative/equivocal because of the band indicated by the arrow. Sample T4 is a characteristic separation of a negative sample, whereas T2 and T3 represent positive samples. Separations performed in FC-coated capillaries and linear PA-coated channels using 10 g/L HEC in 1x TBE. Other conditions as stated in text.

Analysis of the IGH gene rearrangement products via microchip electrophoresis also yielded profiles similar to those seen for the CE analysis (Fig. 6 ). Electropherograms B2 and B3 are representative of negative samples, whereas electropherograms B1 and B4 are characteristic of positive samples. A slight reduction in resolution can be seen in the electropherograms for sample B1 with the microchip (Fig. 6B ) compared with the capillary (Fig. 6A ) separations. However, it is improbable that this slight reduction will influence the diagnostic capacity.



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  		Figure 6. IGH gene rearrangement product separations in a capillary and electophoretic microchip.

Separation of samples labeled in Fig. 1BUp via capillary (A) and microchip (B) electrophoresis. Samples B1 and B4 are positive, and samples B2 and B3 are negative. Separations performed using 10 g/L HEC in 1x TBE in FC-coated capillaries and PA-coated channels. Other conditions as stated in text.

Evaluation of PVP as a sieving matrix for DNA separations in bare silica channels.
PVP, a polymer that has been shown to function as a sieving matrix for DNA and also a dynamic coating for bare silica capillaries (27), was evaluated for DNA separations in bare silica channels. Dynamic coatings can be added to the separation buffer to temporarily adhere to, and therefore deactivate, the silica surface. This is in contrast to traditional capillary coatings, such as the FC and PA coatings, that are covalently bound to the capillary surface and require time-consuming coating processes. Initially, this dynamic coating/sieving matrix polymer was tested in a 37 cm x 50 µm bare silica capillary for comparison with the standard HEC/FC-coated capillary approach. Fig. 7 A shows that the PVP/bare silica approach for separating the DNA fragments (HaeIII digest of pBR322) provides effective resolution of DNA in less than 15 min (Fig. 7A ) in addition to achieving single-base resolution as evidenced by the separation of the 123- and 124-bp fragments.



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  		Figure 7. Separation of a DNA marker, HaeIII digest of pBR322, using PVP in uncoated capillaries and channels.

(A), capillary separation of the DNA marker by 20 g/L PVP in 1x TBE in a bare silica 37 cm x 50 µm capillary. (B), separation on a bare silica microchip using 20 g/L PVP in 1x TBE.

Because of the potential advantages of a dynamic coating sieving matrix and the positive results obtained with the capillary format, PVP was tested as a DNA sieving polymer for microchip electrophoresis. Fig. 7BUp shows the separation obtained in a 4.2-cm channel. The resolution obtained with the microchip was clearly inferior to that obtained with HEC on the microchip with the same channel length (Fig. 4AUp ). Despite the lower resolution, the IGH gene rearrangement products were analyzed with the PVP system to test applicability to a clinically relevant analysis (Fig. 8 ). As shown, those samples that were positive (B1 and B4) and negative (B2 and B3) for B-cell clonality are clearly distinguishable. Similar profiles are observed for the negative samples (B2 and B3) when compared with the capillary and microchip separations in HEC (Fig. 6Up , B2 and B3). However, the positive sample (B1 and B4) profiles display multiple peaks that were not observed in the HEC systems (Fig. 6Up , B1 and B4).



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  		Figure 8. Separation of IGH gene rearrangement products using 20 g/L PVP in 1x TBE on an uncoated microchip.

Samples correspond to those in Fig. 1BUp and Fig. 6Up . Samples B1 and B4 are positive, and samples B2 and B3 are negative.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many clinical diagnostic assays rely heavily on molecular biology techniques for identification of nucleic acid sequences indicative of disease. The cornerstone of many such analyses is the amplification of target sequences via PCR; such is the case for the diagnosis of lymphoproliferative disorders. Although dependent on the type of T-cell lymphoproliferative disorder, PCR/slab gel electrophoresis methods on average detect 75–90% of the clonal populations that are detected by Southern blot (20)(22)(28). Validation studies in the Department of Pathology at the University of Pittsburgh Medical Center have demonstrated that the IGH PCR method used in these studies shows a clonal band in 70% of the cases demonstrating rearrangement by the gold standard, Southern blot analysis, with a probe specific for the heavy chain joining region. Despite the lower detection rates of the PCR/slab gel assay, PCR methods offer the advantages of decreased turnaround time, the ability to analyze a wider range of specimen types and amounts, and in some cases, greater sensitivity for detection of smaller tumor burden. As a result of these advantages, the PCR/slab gel assays are commonly used, as evidenced by their routine use in many clinical laboratories including the Molecular Diagnostics Laboratories at both the Mayo Clinic and the University of Pittsburgh Medical Center. However, substantial room for improvement in the methodology clearly still exists, especially with respect to expediting the separation portion of the assay.

Although slab gel electrophoresis is clearly the established method for interrogation of PCR-amplified DNA fragments, capillary-based electrophoresis has been gaining momentum since its introduction at the beginning of this decade as an alternative format (3). Although not performed in this work, sensitivity comparisons have been performed and have shown that sensitivity is equivalent between the CE and slab gel techniques (24). Both electrophoretic techniques (capillary and slab gel) can detect a positive control diluted 1:100 in thymus-derived DNA but failed to detect a 1:1000 dilution. Microchip electrophoresis, a miniaturized format of CE, has begun to attract attention as a higher efficiency embodiment of CE (14)(15) where the potential for parallel processing and integration of other chemistries can be realized (9)(23).

Oda et al. (24) achieved TCRG gene rearrangement product separation by CE in 17 min using 10 g/L HEC in a 47 cm x 50 µm DB-17-coated capillary at 260 V/cm. The results given in Fig. 2AUp parallel these results and show that the amplified products of TCRG genes can be separated in less than 15 min via CE in FC-coated µ-SIL-FC capillaries, which were found to provide better reproducibility and longer lifetimes for DNA analysis than DB-17-coated capillaries. The resolution obtainable with the conditions described in the original study (24), although adequate for the T-cell analysis, was not readily applicable to B-cell analysis. The smaller fragments (80–140 bp) amplified with B-cell gene rearrangements (see Fig. 1Up ) require higher resolution conditions in this region than conditions that provide adequate separation of the primers and product.

Perhaps most important is the demonstration that the optimized conditions developed with CE could be translated directly to the microchip for DNA analysis. In the capillary system, FC-coated µ-SIL-FC capillaries were used because of their adequate stability and reproducibility; however, microchips with FC-coated channels are not commercially available. The method of Hjertén (26), which provides a relatively stable PA coating of the silica surface, is one of the most common methods utilized for silica deactivation. Using this methodology, comparison of the microchip-based electrophoretic separation with separation in the capillary clearly demonstrates a substantial decrease in analysis time with a negligible loss of resolving power. In fact, a standard approach for calculating resolution (29) showed that resolution on the microchip was higher for the larger fragments (increased ~28% for 267- to 587-bp fragments) but lower for the shorter fragments (decreased ~24% for 184- to 234-bp fragments; decreased ~55% for 51- to 104-bp fragments). Despite these minor changes in resolving power (which can be accounted for by differences in the silica surfaces, the surface coatings, and/or discrepancies in the applied voltages), it is clear that the same diagnostic information can be extracted from both systems. This is exemplified by the ability to detect the suspicious band/peak (fragment size ~140 bp on the gel; indicated by the arrow in Fig. 5Up ) in sample T1, which led this sample to be classified as negative/equivocal. This is of seminal importance when envisioning how multiplex microchip electrophoresis (parallel analysis of many samples) could impact throughput and turnaround time in molecular diagnostics. The benefits (e.g., in this particular case, of reducing analysis time from 2.5 h with slab gel electrophoresis to 160 s with microchip electrophoresis with no apparent compromise in the quality of the information provided) are obvious.

Although the performance (resolution) of the microchip and capillary systems were comparable in terms of extracting diagnostic information, the robustness of the two platforms differ at this point. Effective analysis of the PCR samples via CE required only a simple 1:10 dilution of the PCR product with Tris/EDTA buffer. This is consistent with the previous observations that the salt concentrations typically associated with standard PCR mixtures do not to cause extensive problems with capillary-based separations (30)(31)(32). In fact, studies have shown that PCR product injected directly into the capillary for CE analysis does not affect the quality of the separation (32). This contrasts results with the PA-coated microchip, which appeared to be extremely sensitive to the high salt concentrations of the PCR mixture. This is in agreement with previous studies in which either desalting (33)(34) or extensive dilutions (14) were necessary sample pretreatment steps. The loss of electrophoretic functionality is probably the result of deterioration of the channel coating because exposure to salt led to loss of efficiency (broader peaks) and increased migration times. Although desalting the samples before microchip analysis circumvented this problem, this presents a disadvantage because it burdens the test protocol with additional steps that eventually impact the cost of the test. However, it is noteworthy that this problem may be resolved by using different surface passivation approaches. Hofgärtner et al. (35) have recently shown that use of chemistries that provide a more stable surface coating can obliterate the salt-sensitive nature of microchip electrophoresis. In that study, the successive injection of several hundred PCR samples that had not been desalted or extensively diluted was achieved without impacting the quality of the separation.

Under the conditions described in this report, replicate analysis of the same sample or of many different samples showed reproducible profiles, provided that the PCR samples were desalted before analysis. However, an extensive statistical analysis of reproducibility needs to be performed to evaluate the robustness of this electrophoretic platform. It is clear that the reproducibility of DNA electrophoresis and robustness of the microchip will be directly related to how well the microchannel surface is passivated. As far as covalent modification of the surface is concerned, the PA coating appears adequate but salt sensitive, and improved deactivation chemistries (35) are likely to improve on this. Perhaps the most convenient approach will be the use of a polymer that not only provides sieving of the DNA but also deactivates the surface in a dynamic fashion. This is attractive because most covalent coatings that deactivate silica surfaces are difficult to produce uniformly and reliably and have limited lifetimes. The virtues of PVP (27)(36) and poly(ethylene oxide) (37) as polymers that can dynamically deactivate silica surfaces and provide a sieving matrix for capillary DNA separations have been proclaimed. We show, for the first time, that the microchannel can be dynamically deactivated with PVP (Mr 360 000) and PCR-amplified DNA resolved with the same polymer. The resolution was poorer than that observed with HEC, possibly because the surface of the microchannel was rougher than in a capillary (38) and, hence, not as effectively deactivated. The multiple peaks observed in the IGH gene rearrangement products (Fig. 8Up ) were not seen in the HEC/FC capillary system (Fig. 6AUp ), but similar multiple peak profiles were obtained for the same samples separated in bare silica capillaries using PVP. Despite the multiple peak profile, which is currently under investigation, the use of PVP as a dynamic coating/sieving matrix for DNA separations in uncoated electrophoretic microchips under unoptimized conditions allows diagnostic information about IGH gene rearrangement to be obtained. Although there are idiosyncratic issues to be resolved, these results illustrate the potential of dynamic coatings that also function as sieving matrices for simplifying the use of uncoated electrophoretic microchips for molecular diagnostics.

In conclusion, slab gel, capillary, and microfabricated chip electrophoresis have been used to analyze TCRG and IGH gene rearrangements, molecular diagnostic assays used to detect T- and B-cell lymphoproliferative disorders. The same information obtained from the slab gel and CE was extracted by the electrophoretic microchip, except with an analysis time that was dramatically reduced. Decreasing the time needed for electrophoresis from as long as 2.5 h on the slab gel to 15 min on the capillary and finally to 160 s on the electrophoretic microchip has obvious implications for diagnostic testing. Although the microchip separations primarily used a PA channel coating and HEC for separation of the DNA fragments, the use of PVP as a dynamic coating/sieving matrix for DNA separations was shown to be feasible. This approach may eliminate the time-consuming channel coating process and irreproducibility problems accepted with silica coatings, allowing for minimal preparation and rapid analysis. Inherent in considering electrophoretic microchip technology as an analytical tool in clinical diagnostics will be its comparison with conventional methods. In this report, microchips are shown to reduce electrophoretic analysis time by 60-fold while maintaining full diagnostic capacity. This is the first step toward accepting this technology as a new paradigm in diagnostics.


   Acknowledgments
 
This work was funded in part by the National Cancer Institute (Grant 1R21CA78865-01 to J.P.L.), the Lubrizol Corporation (to N.M.), and ACS Analytical Division Summer Fellowship sponsored by the R.W. Johnson Pharmaceutical Institute (to N.M.). We would like to thank Dr. Zhili Huang, University of Pittsburgh, for designing, building, and writing the Labview program for the microchip high-voltage power supply, and Beckman Coulter for instrumentation.


   Footnotes
 
1 Nonstandard abbreviations: CE, capillary electrophoresis; TCR, T-cell receptor; IgH, immunoglobulin heavy chain; LIF, laser-induced fluorescence; TBE, Tris-borate-EDTA; HEC, hydroxyethylcellulose; PVP, polyvinylpyrrolidone; FC, fluorocarbon; and PA, polyacrylamide.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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