Challenges In Genomic Analysis

Plasmid Preparation for DNA Sequencing

Many procedures have been developed over the years for isolation of bacterial plasmids.8 Further, several proprietary methods also give satisfactory results. One aspect that nearly all of these methods share is that they involve three basic steps: growth of bacteria, harvesting and lysis of the cells, and purification of the plasmid. All include centrifugation or several treatments of the colonies that are labor intensive and difficult to automate and to interface with CAE. Some chromatographic columns (size exclusion, ion-exchange, high-performance membranes, hydrophobic interaction) have also been developed to avoid the use of centrifugation. All these methods, which may meet the stringent quality criteria for gene therapy, involve high cost and intensive labor. Another complication is that reagents used in the purification of plasmids, such as ethanol and SDS, might become interferences to the subsequent cycle-sequencing reaction.

Alternative methods, which require only heat-induced lysis of cells in bacterial colonies, also exist.9,10 Centrifugation or vigorous vortex is still needed to isolate cell debris. The resulting lysate is then used as the template in cycle sequencing using labeled primers. Even though the performance and the ruggedness demonstrated so far by this method are still inferior to that of the standard protocol, the method shows promise for significant savings in time and cost. In fact, a microwave protocol similar to above procedure has already become the preferred method for purification of double-stranded DNA at the Washington University Genome Sequencing Center.11 In its method, the growth of bacterial cultures and subsequent DNA isolation took place in the same 96-well block and no further purification of DNA, by precipitation or other means, was necessary. The 96-well blocks can be reused indefinitely provided they were cleaned between uses. Furthermore, the lysis solution was easily made and was stable at room temperature for a minimum of 3 months, allowing liter-quantity batches to be made and stored. The cost was estimated to be $0.03/sample. This compared very favorably with the cost of commercial preparation methods, which can be as high as $1/sample. Using this approach, the average high-quality sequence length was 427 bases, while 70.52% of the sequences had at least 400 bases of high-quality data.

DNA Ladder Purification and Sample Injection

Capillary gel electrophoresis (CGE) is an attractive technique for DNA analysis because the narrow-bore, gel-filled capillaries provide high-speed, high-resolution separations, as well as automated gel and sample loading. The use of CGE for DNA

sequencing was first demonstrated in 1990, when sequencing separations of ~350 bases were obtained in cross-linked gels in ~80 min.12 Much progress has been made in the past decade, and sequencing read-lengths of more than 1000 bases can now be obtained using replaceable gels.1314 In fact, CGE has eliminated the bottleneck involved in the separation of the DNA ladder produced by the Sanger reaction and has become the key technology for DNA sequencing.

Compared with slab-gel electrophoresis, the sample in CGE is injected into the separation capillary instead of being loaded into wells. Therefore, dye-labeled DNA fragments must compete with ions in the sample matrix when they enter the capillary. It is well documented that the performance and reliability of DNA sequencing by CE is sensitive to the quality of the DNA sample due to the employment of elec-trokinetic injection. Residual salt and dideoxynucleotides in the sequencing sample cause discrimination against DNA because they have higher mobilities. As a result, large variability in signal strength is often observed in CGE. Another problem associated with sample purification is the rehybridization of the single-stranded DNA injected. This causes the mobilities to change and makes base calling difficult.

The benefit of stringent sample purification has been demonstrated before.1516 A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts to below 10 |M in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10- to 50-fold compared to traditional cleanup protocols. Using M13mp18 as a template, the resulting purified single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. A systematic study to determine the quantitative effects of the sample solution components, such as high-mobility ions (e.g., chloride and dideox-ynucleotides) and template DNA, on the injected amount and separation efficiency revealed that, in the presence of only 0.1 | g of template in the sample (one third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentrations (~5 |M), the separation efficiency decreased by 70%. The deleterious effect of template DNA on the separation of sequencing fragments was not observed in slab-gel electrophoresis because it was suppressed in the presence of salt at a concentration above 100 |M in the sample solution. The latest results showed that read length up to 1300 bases (average 1250) with 98.5% accuracy can be achieved in 2 h for single-stranded M13 template.17 Thus, the purified DNA ladder dramatically improved the result but at the expense of high cost and manual manipulation.

Swerdlow et al.18 first tried to perform DNA sequencing with unpurified DNA sequencing samples. They used a method called base stacking, which allowed direct injection of unpurified products of dye-primer sequencing reactions onto capillaries without any pretreatment. Briefly, on-column concentration of DNA fragments is achieved simply by electrokinetic injection of hydroxide ions. A neutralization reaction between these OH- ions and the cationic buffer component Tris+ results in a zone of lower conductivity, within which field focusing occurs. Without base stacking, a drastic loss in signal was observed for the crude samples. This method can generate separation resolution of at least 0.5 up to 650 bp. The signal strength was excellent relative to conventional injection of highly purified samples. Furthermore, no significant degradation of the capillary performance was observed over at least 20 sequencing runs using this new sample injection method. One shortcoming of this method is that it did not yield satisfactory results with dye terminator chemistry due to the interference of unreacted dye terminators.

Preparation of Biological Samples for PCR

It is interesting that a new PCR method, FoLT (formamide low temperature) PCR, has been developed for reactions directly from whole blood. Formamide solubilizes blood cells and frees the DNA for amplification An important finding was that an alternative DNA polymerase, Tth polymerase, was less sensitive than Taq polymerase to the presence of proteins in blood. All these make PCR directly from blood possible.19,20

Although blood can be used directly in PCR, there is clearly a need for simpler, noninvasive, and more cost-effective means of sample collection, DNA extraction, and genetic diagnosis in general. There are several disadvantages of using blood. First, blood collection can be very inconvenient, because genetic testing often involves analysis of multiple family members. Furthermore, drawing blood can be uncomfortable for the patient and, most important, the handling of blood samples can increase the chances of infection by blood-borne pathogens such as HIV and hepatitis. To date, a variety of alternative sources of DNA have been used for genetic testing including finger-prick blood samples, hair roots, as well as the use of cheek scrapings and oral saline rinses as a means of collecting buccal epithelial cells. The oral saline rinse is perhaps the most extensively used nonblood-based sampling technique. However, it still involves liquid sample handling and requires an additional centrifugation step to spin down the cells, which is difficult to automate and interface with subsequent analysis.

By avoiding centrifugation, a simpler method has been developed and validated by using just swabs and brushes.21 The buccal cells were collected on a sterile brush by twirling the brush on the inner cheek for 30 s. Although still requiring a neutralizing step later, this method is generally easy and very reliable. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of the results for blood and cheek-cell DNA. The success rate of PCR amplification on DNA prepared from buccal cells was 99%. This method has also been used to analyze DNA for genetic polymorphism by matrix-assisted laser desorption/ionization mass spectrometry.22

Total System Integration

Numerous endeavors have been made in developing robotic workstations to perform sequencing reaction, purification, preconcentration, and sample loading. Although robotics has shown advantages in repetitive operation with high precision, the adaptation to highly multiplexed capillary array separation interface suffers from many incompatibilities in terms of the total reaction volume, purification by centrifugation, and sample injection. Online microfluidics systems based on either capillaries or microchips hold promise for the next generation of totally automated DNA sequencers.

Capillary Microfluidics Systems

In capillary microfluidics,23 dye-labeled terminator cycle sequencing reactions are performed in a 250 |im i.d. fused-silica capillary, which was placed into a hot-air thermal cycler. After PCR was completed, the reaction mixture was transferred online to a size-exclusion column to purify the reaction product from the unreacted dye terminators. The purified product was then injected through a cross into a gel-filled capillary for size separation. This system was closed and the operation was reliable since no moving parts were involved. Cleaning of the system with 0.1 M NaOH was required to remove cross-contaminants before reuse. The sequence could be called from 36 to 360 bases with an accuracy of 96.5% using in-house software. By manual editing, the accuracy improved to 98% for 370 bases. Later on, a multiplexed system based on above scheme was developed in which eight DNA sequencing samples could be processed simultaneously starting from template to called bases.24 The major achievement in the instrument was the use of freeze/thaw switching valves instead of rotary valves, which were unsuitable for multiplexed systems due to their size. For all eight processed samples, sequences could be called up to 400 bases with an accuracy of 98%. PCR analysis directly from blood was also demonstrated with a similar flow management concept.25

Another fully integrated single capillary instrument comparable in design has also been designed and prototyped.26 The reaction was performed inside Teflon tubing. The purification and separation columns were interfaced through a simple T-connector instead of a cross. The instrument was reliable and fast, performing PCR reaction cycling, purification, and analysis all in 20 min. Adaptation of the instrument prototype for separation of DNA-sequencing reactions was described; cycle sequencing and electrophoresis of a single lane were complete in 90 min with base calling to beyond 600 bases.

Miniaturization of the online system will reduce the cost of DNA sequencing substantially below current levels because only 1/100 of the reagent is required for actual CE. Soper et al.27 developed a miniaturized solid-phase cycle sequencing reactor coupled with CE. The nanoreactor consisted of a fused-silica column with a total volume of 62 nl. Biotinylated DNA template was bonded to the surface by biotinstrepavidin-biotin linkage. The main disadvantage of this scheme is the loss of template surface coverage. One solution for reactivation of the nanoreactor could be through adding fresh streptavidin and new biotinylated target DNA. The read length for a single-color run was approximately 450 bases. The system is considered amenable to automation even though there is still manual operation in the published design.

Online Microchip Systems

Microchips provide a new platform for integration with unique electro-osmotic pumping and nonmechanical valves. A true nano-total analysis device was developed by Burns et al.28 who used microfabricated fluidic channels, heaters, temperature sensors, and fluorescence detectors to analyze nanoliter-size DNA samples. The device is capable of starting with 100 nl reagent and DNA solution, online mixing, amplifying or digesting the DNA to form discrete products, and separating and detecting those products in microfabricated channels. No external lenses, heaters, or mechanical pumps are necessary for complete sample processing and analysis. The components have the potential for assembly into complex, low-power, integrated analysis systems at low unit cost.

Microchips still need to overcome some technical difficulties before they can be routinely utilized. These include solvent evaporation and interface with the outside bioanalysis laboratory where the common working volume is |l. Litborn et al. described using a closed humidity chamber to address the problem of solvent evaporation.29 Later, they reported an improved technique for performing parallel reactions in open, 15-nl volume, chip-based vials. The evaporation of solvent from the reaction fluid was continuously compensated for by addition of solvent via an array of microcapillaries. Their results showed that the concept for continuous compensation of solvent evaporation should be applicable to reaction volumes down to 30 pl.30

There are other approaches to avoid the problem of solvent evaporation. Soper developed a hybrid system that coupled nanoliter sample preparation to PMMA (polymethylmethacrylate)-based microchips.31 Unlike standard sample preparations that are performed off-chip on a | l scale, true integration was demonstrated at nl volumes. An integrated system for rapid PCR-based analysis on a microchip has also been demonstrated recently.32 The system coupled a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel elec-trophoresis platform. This configuration allowed fast (~1 min/cycle) and efficient DNA amplification on-chip follo- wed by electrophoretic sizing and detection on the same chip. An unique on-chip DNA concentration technique based on adsorption and desorption has been incorporated into the system to reduce analysis time further by decreasing the number of thermal cycles to 10 cycles, or 20 min for DNA amplification and subsequently detection.

Capillary Array Instruments

In 1992, Mathies's group developed this approach to address the throughput requirement of genomic analysis.6 They constructed a confocal fluorescence scanner and demonstrated DNA sequencing in 25 parallel capillaries. Since then, additional improvements in optical design and separation matrixes have made the commercialization of CAE sequencers possible. Today, there are four commercial versions of CAE instruments. PE Biosystems has developed the ABI PRISM 3700 DNA analyzer. This 96-capillary array instrument is based on the approaches of Kambara8 and Dovichi.7 In this instrument, DNA sequencing fragments are detected in a sheath flow and spectrally resolved using a concave spectrograph and a cooled charged coupled device (CCD) camera. Bare capillaries are used with dynamic coating, which is stable for more than 300 runs. The turnaround time is roughly 2.6 h with 600 bp in 120 min. The Molecular Dynamics instrument MegaBACE 1000 is based on confocal detection after Mathies's group.6 A microscope objective is used to focus the laser light inside the capillaries and, at the same time, collect the emitted light from the center of the column. The lifetime of the instrument may be limited by mechanical stress of moving the scanner when fast sampling rates are required. The system uses linear polyacrylamide (LPA)-coated capillaries, which are stable for 200 runs. The average sequencing data is 500 bp and the turnaround time is less than 2 h. Beckman Coulter has entered the market with an 8-capillary array design, the CEQ 2000 DNA analysis system. The optical design of this instrument is similar to that of Molecular Dynamics except that separate excitation and emission paths are used. It features four-color IR dideoxy-terminator chemistry. On-column detection is the approach implemented in the SpectruMedix instrument that is based on our work.33 The laser beam crosses all 96 capillaries after the laser is expanded by a cylindrical lens. The fluorescent light is collected at right angles from the laser axis and detected by a CCD camera. Because no moving parts are involved in detection, the optic design is very rugged. Bare fused-silica capillaries are used with dynamic coating with a hydrophobic polymer. The turnaround time is 2 h with average base calling of 500 bp. The SpectruMedix system is also employed in the new 16-capillary ABI 3100 and 96 capillary 3710 sequencers.

Alternatives in system design mainly concern illumination and detection. Kambara's group34 further tested side illumination with detection on column. For this design, the number of capillaries in an array is generally limited by laser-power attenuation along the array due to reflection and divergence. They overcame these problems by placing the capillaries in water and adding glass-rod lenses between the capillaries. As a result, up to 45 capillaries could be simultaneously irradiated with a single laser beam and the fluorescence from all the capillaries could be detected with high sensitivity. Quesada and Zhang35 took another approach for a multiple capillary instrument by the use of optical fibers for illumination and collection of the fluorescence in a 90° arrangement. A subsequent version of this instrument utilized cylindrical capillaries as optical elements in a waveguide, where refraction confined a focused laser beam to pass through 12 successive capillaries in a flat parallel array.36 However, larger capillary arrays are limited by the refractive effects that spread the light along the length of the capillaries.

Handling more than 96 capillaries is very challenging and several groups have attempted to address this problem by modifying existing designs. Dovichi's group used sheath-flow detection and a novel two-dimensional arrangement that can hold up to 576 capillaries.37 A prototype 384-capillary array electrophoresis instrument has also been developed for higher throughput analysis by SpectruMedix. Their instrument design is based on the 96 capillary platform with a redesign in the camera lens. Mathies and coworkers have also continued to push the limit of the confocal system. They have developed a system with the capillaries aligned in a circular array. The microscope objective spins inside a drum, illuminating the capillaries one at a time. They have shown sequencing data from 128 capillaries, but a larger number of capillaries could be easily accommodated in this geometry.3839

A totally different platform to perform CAE is the microchip. The first demonstration of CAE in microchip was by Mathies's group for genotyping.40 A microplate that can analyze 96 samples in less than 8 min was produced by bonding 10 cm diameter micromachined glass wafers to form a glass sandwich structure. The microplate had 96 sample wells and 48 separation channels with an injection unit that permitted the serial analysis of two different samples in each channel. An elastomer sheet with an 8 x 12 array of holes was placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner as described above. An SNP typing assay has also been developed and evaluated in a microfabricated array electrophoresis system.41 That study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high-speed fluorescent SNP typing.

DNA sequencing on the microchip array is very interesting and challenging. Early studies of single channel on a chip required channel lengths comparable to capillaries. Making many turns in a chip also proved to be deleterious to separation performance. It also implies that a larger-diameter chip is needed for DNA sequencing.42 Recent results show that there is still much room for improvement. Liu et al.43 demonstrated DNA sequencing by 16 channel CAE in a microchip format. Samples are loaded into reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescence detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. This demonstrates the potential of the microchip as the next generation CAE platform.

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