microfabrication and array technologies for dna sequencing and diagnostics

E L S E V I E R

Genetic Analysis: Biomolecular Engineering 13 (1996) 151-157

NENETIC ALYSIS

Blomolecular Engineering

Microfabricalfion and array technologies for DNA sequencing and diagnostics

Maryanne J. O'Donnell-Maloney a,*, Daniel P. Little b

aSequenom Inc., 101 Arch Street, Boston, MA 02110, USA bSequenom Inc., 11555 Sorrento Valley Rd., San Diego, CA 92121, USA

Received 15 August 1996; revised 20 October 1996; accepted 24 October 1996

Abstract

It is now possible to miniaturize numerous 'macroscale' processes and develop microfabricated devices to replace conventional equipment. Such advances have lead to the development of arrays of immobilized oligonuceotides useful for basic research, diagnostic studies, sequence analysis and a number of novel applications. Additionally, standard laboratory equipment has been redesigned on a microscale level to increase efficiency; many processes have been integrated onto one chip since miniaturization can be readily achieved. © 1996 Elsevier Science B.V. All rights reserved

Keywords: Microscale; Immobilized oligonucleotides; Miniaturization; Diagnostics

I. Introduction

The ability to miniaturize a known process enables faster analysis time, less waste of costly reagents, easier manipulations and the prospect of massive parallelization; therefore, numerous molecular biological and bio- chemical procedures are being re-examined in the hopes of miniaturizing the scale. Such miniaturization not

* Corresponding author. I Biochip Array Technology and Microfabrication Conferences

held on March 18-19, 1996 in Marina del Rey, California. The conferences were organized by International Business Communica- tions.

z Michael J. Heller, PhD (Nanogen); M. Allen Northrup, PhD (Lawrence Livermore National Laboratory); J. Wallace Parce, PhD (Caliper Technologies); Arnab K. Mallik, PhD (Genometrix); Robert S. Matson, PhD (Beckman Instruments); Lance Fors, PhD (Third Wave Technologies); Thomas Theriault, PhD (Combion); Snezana Drmanac, MD (Hyseq); Paul N. Rys, PhD (Mayo Clinic and Foun- dation); Carol A. Dahl, PhD (National Institutes of Health); Uwe R. Muller, PhD (Vysis); Kenneth L. Beattie, PhD (Houston Advanced Research Center); Rolfe C. Anderson, PhD (Affymetrix); David B. Wallace, PhD (MicroFab Technologies); Yu-Hui Rogers, MS (Molecular Tool, L.L.C.); Timothy Woudenberg, PhD (Perkin- Elmer).

1050-3862/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S1050-3862(96)00166-0

only improves current applications but has lead to a number of novel applications. A plethora of research is being directed towards microfabrication as was evidenced during two recent conferences ] . The numerous presenters 2 and participants shared the advances that have taken place recently.

A large portion of the conference was dedicated to the development of ordered arrays of oligodeoxynucleotides covalently attached to a solid surface which may be used for multiplexed hybridization assays and sequencing by hybridization (SBH). In a typical array hybridization assay, a sample of unknown D N A is applied to the array and the hybridization pattern is analyzed to produce many short bits of sequence information simultaneously. SBH and related array hybridization methods should expedite the daunting task of sequencing the entire human genome by greatly increasing the rate of D N A sequence acquisition and may lead to a number of diagnostic assays. Since SBH and other hybridization assays are already producing sequence information and diagnostic tests, there is a great need for rapid yet robust ways to synthesize the arrays. The numerous steps involved in performing a hybridization assay were discussed in detail at the con-

152 M.J. O'Donnell-Maloney, D.P. Little / Genetic Analysis: Biomolecular Engineering 13 (1996) 151-157

ferences, including chemical attachment of a probe to a surface, physically making a densely packed set of elements on a surface, hybridization conditions on a surface, hybridization detection on a surface, and integration of all these processes.

As well as hybridization technology, many other molecular biological processes are being investigated in anticipation of miniaturization. The Biochip Array Conference chairperson Michael Heller (Nanogen, San Diego, CA) introduced the concept of integrated micro- machined devices; the challenging task of integrating multiple components into a single device at a microscale level is not only currently possible but proving to be quite successful. For example, a number of speak- ers presented data from miniaturized PCR devices, miniaturized integrated PCR/transcription/fragmenta- tion systems and miniaturized diagnostic tools.

2. Synthesis of miniaturized arrays

The actual techniques employed to make densely packed arrays of DNA attached to a solid surface were discussed in detail. In the past, DNA was commonly attached to a surface through non-covalent bonds or through non-specific UV crosslinking of the bases to the surface; currently, however, site-specific covalent attachment of oligodeoxynucleotides has become the method of choice. The two different modes of covalently attaching DNA to a surface are, post-synthetic attachment and in situ synthesis. The former method involves standard automated synthesis of oligodeoxynucleotides, removal of the strands from the support, and chemical immobilization at specific sites on the surface of a chip. In the latter method, the desired oligodeoxynucleotide is synthesized directly on the support that is to be used for subsequent analysis. In addition to the chemistry that is employed to immobi- lize oligodeoxynucleotides on a surface, one must consider the actual manner in which numerous spots are deposited in a microscopic space on a surface, thus, another recurring topic throughout the conference was the use of microdispensing units such as robotic pin tools and piezoelectric ink-jets.

2.1. Chemical attachment of DNA to solid surfaces

2.1. I. Post-synthetic attachment o f DNA to surfaces Kenneth Beattie (Houston Advanced Research Cen-

ter, Woodlands, TX) described a few different ways to covalently attach DNA to silicon surfaces. Beattie pointed out that previous efforts to condense the pri- mary amine of a 5'- or T-terminally modified oligodeoxynucleotide with an epoxy-functionalized silicon surface produced densities of approximately 100 fmol DNA/mm 2 surface, but the reaction proceeded

rather slowly and required the use of a humidity chamber. More recently, the researchers have directly coupled a 3'-propanolamine-derivatized oligodeoxynucleotide to an unmodified silicon dioxide surface. This reaction produced binding densities similar to the epox- ide chemistry, however the reaction is simpler since it lacks a modified surface and proceeds more quickly. With both chemistries, dimpled surfaces were employed in which the oligonucleotide solution was placed only in the wells. Densely packed arrays of 240 (10 x 24) and 800 wells (20 × 40) were made using robotic pin spotting; however to make arrays of 2592 wells (36 x 72), ink-jet technology was employed as will be discussed in a later portion of this article. Beattie also reported on the covalent attachment of DNA to 'flow-through genosensors' which are porous silicon dioxide surfaces, replacing the two-dimensional surfaces commonly used. The greater surface area per unit cross section of these genosensors produces approximately 100 fold increase in the density of bound oligonucleotide compared to 'flat' surfaces and improves the detection sensitivity. Additionally, a solution flowing through the pores rapidly encounters the surface tethered oligonucleotide since the distance to the surface is quite short thus reducing hybridization times. Evaporation can be elimi- nated since the liquid-filled pores may be covered while still allowing a reaction to continue in solution within the pores. Because of these advantages, it is possible to capture hybrids from a dilute solution and to hybridize denatured double-stranded PCR fragments without prior isolation of a single strand. The flow-through pores also enable a denaturing solution to be rinsed through them, thus hybridized oligonucleotides may be recovered for further analysis.

2.1.2. In situ synthesis There are essentially two different ways to synthesize

DNA in situ on a surface to produce a set of spatially distinct and highly diverse chemical products. The first method employs standard phosphoramidite chemistry except for the linker that is used to attach the 3' base to the support. The second method combines solid phase chemistry, photolabile protecting groups, and pho- tolithography. Robert Matson (Beckman Instruments, Fullerton, CA) described disposable oligonucleotide array 'genosensors' that are synthesized by the southern array maker (SAM) using standard automated phosphoramidite chemistry. The support employed is a clear polypropylene film which is thermoplastic, moldable, not easily fractured and resistant to solvents used in DNA synthesis. The surface of the film is modified using radiofrequency plasma discharge in an ammonia atmosphere to place an amino group on the surface. The aminated polypropylene is placed in the SAM synthesizer, and standard phosphoramidite chemistry is directed in 64 distinct and independent channels to

M.J. O'Donnell-Maloney, D.P. Little / Genetic Analysis: Biomolecular Engineering 13 (1996) 151-157 153

produce 64 different oligonucleotides. Most commonly, the genosensor takes the form of a 'dipstick' with dimensions 0.5 x 8 cm with 64 0.5 mm channels. For quality control, the same oligonucleotide was synthesized in all 64 channels, the oligonucleotides were cleaved from the support, each channel's product was analyzed and comparison revealed 90-93% accuracy. In a similar experiment, ~Ln oligonucleotide was cleaved from the support and analyzed showing approximately 98% purity (2% failed sequences). The amount of oligonucleotide synthesized on the polypropylene support is 1-3 pmol/mm 2, however, only 0.1-1% is available for hybridization. The genosensors have been used in hybridization studies on cystic fibrosis, short tandem repeats screening, and RzLs oncogene mutational analysis.

Thomas Theriault (Combion, Pasadena, CA) described another mode of automated in situ DNA array synthesis on a two dimensional surface using standard phosphoramidite chemistry. In this mode, a piezoelectric ink-jet dispense[ is employed to place discreet droplets of a reagent on the surface of the chip. Two different array-making schemes were described by Theriault. In the first, a protected linker is attached to the surface in a patterned array, then a deprotecting solution is delivered to specific spots of the array and only the deprotected sites, are coupled to the appropriate base in a subsequent step. These reactions are all done using a single stream of solution from one chem- jet, therefore four sets of steps are necessary to dispense each of the four bases and build all oligonucleotide sites by one base. In a more recent approach, the protecting group on the linker is removed simultaneously from all spots of the array using a multi-jet dispenser. Then multiple jets dispense the appropriate base solution to each site on the array; therefore, it is possible to extend all sites of the array by one base in a single step. The multi-jet dispenser is now used more commonly due to the expedience of time. In order to monitor the stepwise coupling yield, a cleavable linker was placed between the support and the T-base and the cleaved oligonucleotide was analyzed to reveal a stepwise coupling yield of only 91%. As well as the poor coupling yields, a second potential disadvantage results from mixing of adjacent spots due to drop spreading. In order to investigate this concern, an array of 10000 spots in 1 cm 2 was synthesized with fluorescently labeled oligonucleotide probes; analysis using a fluorescence scanning detector showed that the majority of spots possessed discreet edges and were se, parated from their neighbors. Hybridization was also monitored with fluorescently-la- belled sequences and was determined to be approximately 2 fmol DNA/mm 2 surface. In an attempt to increase hybridization, Theriault and his co-workers examined a variety of linker groups between the 3'-base of the arrayed oligodeoxynucleotide and the support

and found that a relatively long linker such as a polyethylene glycol resulted in the highest hybridization efficiency. The researchers are now attempting to imple- ment genetic diagnostics and genetic identification on their arrays.

Rolfe Anderson (Affymetrix, Santa Clara, CA) reported on the second strategy to synthesize oligodeoxynucleotides in situ. In this method, a protected linker is attached to a two-dimensional surface, light is employed to cleave these photolabile protecting groups in a specific pattern, then the entire surface is exposed to a phosphoramidite, but coupling only takes place where the protecting group has been removed. Masks control which regions of the array are exposed to illumination and thus are activated for chemical coupling. The cycles continue until a diverse set of spatially defined oligodeoxynucleotides has been synthesized. To date, 64000 oligonucleotides have been synthesized on 1 c m 2. Hybridization to the array is monitored using fluorescently tagged complementary sequences and a confocal fluorescent microscope. Such arrays have been utilized in HIV-1 protease studies in which PCR was performed, the DNA was transcribed into RNA, then the RNA was fragmented and hybridized to the chip for analysis.

2.2. Sample deposition on arrays

When considering the design of a microfabricated array, one must consider the desired spot size of each element, the spacing between the elements and the method of sample delivery to the array. Quite often, the number of elements and spatial distribution prohibit manual deposition, therefore robotic deposition must be used. When robotic deposition is required, there are two possible means of sample delivery, a pin tool or ink-jet technology, both of which were discussed at the Microfabrication Workshop.

2.2.1. Robotic pin tool Uwe Mfiller (Vysis, Downers Grove, IL) discussed

Genosensor-based comparative genomic hybridization for the detection of genetic abnormalities. With a diagnostic genosensor as the goal, Mfiller addressed the numerous challenges of miniaturization and genosensor manufacturing. Ultimately, to make a genosensor with 600-2500 targets per slide, it would be necessary to dispense picoliter volumes and to dispense multiple fluids simultaneously. Manual pipetting was shown to deliver down to approximately 0.2 ktl in 1 mm spots, but this technique is not viable for genosensor manu- facture. Pin transfer was described where nanoliters of liquid were dispensed from the head of a finely manu- factured tip. This technique dispenses more liquid per drop than ink-jetting, but the pin tool may be multiplexed to dispense 384 liquids at once, while ink-jet

154 M.J. O'Donnell-Maloney, D.P. Little / Genetic Analysis: Biomolecular Engineering 13 (1996) 151-157

technology has not been multiplexed to that level, as will be discussed in a later portion of this article.

Kenneth Beattie (Houston Advanced Research Cen- ter, Woodlands, TX) also described the use of a robot adapted with pins to transfer liquid from a microtiter plate to a microscope slide. With such a system, it was possible to transfer approximately 10 nl of liquid on a 1-2 mm pitch onto a flat surface. It was determined that such a deposition of liquid produces a linear application of probes on a surface. More recently, an array of wells have been filled with solution using a similar tool in a 10 x 24 array and in a 20 x 40 array.

2.2.2. Ink-jet technology David Wallace (MicroFab Technology, Plano, TX)

described ink-jet technology from the basics to future applications. Basically there are two different technologies that can be employed, continuous and drop-on-demand. Continuous flow causes fluid to be pumped through a transducer, while a charge electrode and deflection plates directs drops either to the substrate or to a catch basin. Such a system causes the fluid to break up into repeatable drop sizes, but a distinct disadvantage of this system is that droplets must constantly be produced during the operation, thus possibly wasting costly reagents. Drop-on-demand technology, on the other hand, uses a piezoelectric shooter to produce spheres of fluid 25-100 /lm in diameter at a rate of 0-6000 spheres/s. Non-continuous spray and the decreased drop volume (10-500 pl) minimize the waste of expensive reagents. Additionally, as opposed to pin spotting, the accuracy of piezoelectric ink-jet printing is not affected by substrate wetting so the resultant array has more reproducible spot size and shape. For DNA array fabrication, an integrated system of eight fluid microdispensers has been designed and executed. An array of 100 /~m spots on a 300 ¢tm pitch was made using the integrated printing system with two fluorescent dyes. The results revealed discreet, distinct spots in an essentially uniform array. An array of DNA covalently attached to glass was made according to the chemistry of Kenneth Beattie (as described above) using the eight fluid dispensing system with a varied number of drops per spot. Radiolabelled complementary sequences were hybridized to the array and analyzed to reveal a linear relationship between spot volume and hybridization signal.

Thomas Theriault (Combion, Pasadena, CA) also spoke about the application of ink-jet technology to DNA array production. Originally, a single-jet was employed for array synthesis, but currently a multi-jet dispenser is used to print on an entire array in one step, as was described previously. Uwe Miiller (Vysis, Down- ers Grove, IL) touched on the advantages of multiplex- ing ink-jet technology for DNA microfabrication; these advantages include decreased production time over a

single jet, spot reproducibility that is superior to pin spotting, and lower sample volumes that are consumed in array making. However, unlike the pin tool which has been multiplexed 384-fold, such an array of piezo jets represents a significant technical challenge since each jet requires its own piezo element.

3. Applications of microfabricated devices

The nature of sequencing and diagnostics of DNA, which has its information encoded in three billion bits of information of only four forms (A, T, C, G), has inspired significant efforts to develop methods for immensely parallel data acquisition. Concurrently the potential for improvements in speed, cost, and efficiency of analytical and diagnostic instrumentation by miniaturization is becoming well recognized and made more practical by advances in microfabricated silicon technology. Recently, several groups developing improved electrophoresis, electrochemistry and chromatography schemes have reduced these theoretical advantages to practice, and such advances are being incorporated into instrumentation commonly used for biotechnology. Ap- plications of such miniaturized devices and their potential contributions to parallel detection of DNA diagnostic and/or sequencing products are the topic of this section.

3.1. Hybridization diagnostics on a chip

Analyses based on specific hybridization of target DNA to surface bound oligonucleotides is particularly amenable to the rapid parallelization and automated processing, especially if this array is confined to a small surface area. Various DNA diagnostic and de novo sequencing schemes based upon monitoring such hybridization patterns were presented. The biggest de- mands for such arrays are put forth by groups utilizing SBH; here a typical chip may require up to 65 536 different oligonucleotides (all possible 8-mers) attached to a surface. The need to miniaturize such arrays is obvious since PCR reactions or enzyme digestions typi- cally produce volumes on the order of 100 ktl or less of target which must be distributed among the entire array of oligonucleotides.

As pointed out by Arnab Mallik (Genometrix, Woodlands, TX) the development of such chips requires the integration of surface chemistry, electrical engineering and physics for optical detection, computer science and bioinformatics. A high resolution integrated lenseless charged coupling device was presented for the detection of radiolabeled probes on an array; speed improvements ranged from one and two orders of magnitude over gas phase array and X-ray film detection, respectively.

M.J. O'Dgnnell-Maloney, D.P. Little / Genetic Analysis: Biomolecular Engineering 13 (1996) 151-157 155

Snezana Drmanac (Hyseq, Sunnyvale, CA) introduced the 'Super-chip' consisting of an array of 1024 immobilized probes, all possible pentamers, spaced by hydropho- bic strips on the surface of a chip. Upon presentation of a solution containing 1024 labeled 5-mers and a template, the solution phase 5-me~:s are ligated to the support bound 5-mers only in those positions for which a complementary sequence exists in the template. Thus, the same information is obtained by synthesizing two sets (labeled and unlabelled) of 103 5-mers as would be obtained by synthesizing the greater than 106 possible 10-mer probes, resulting in immensely reduced costs of oligonucleotide synthesis and quality control. The com- plex chip described above is only necessary for applications of de novo sequencing; far smaller arrays can be utilized for DNA diagnostics in which the presence or absence of a particular base mutation is being screened. A heterozygous cystic fibrosis exon 11 allele was correctly identified upon its specific binding to a series of four immobilized 7-mers; for the two perfect matches of the 7-mers to the templates, binding and ligating of labeled solution phase 5-mers produced signals far more intense than for the 7-mer to template pairings with a single base mismatch. Normal, mutant, and heterozygote alleles of the common cystic fibrosis exon 10 AF508 mutation could be differentiated in a similar manner.

A second array hybridization-based diagnostic method (genosensor-based comparative genomic hybridization, GCGH) was introduced by Uwe Miiller (Vysis, Downers Grove, IL). GCGH is a means of providing a global view of the genome by testing for the presence of large insertions and/or deletions; specifically, applications for genetic testing by GCGH include single and rare cell analyses relevant to detection of cancer cells in body fluids or fetal cells in maternal blood. For low resolution measurements, a genosensor is contemplated which con- sists of a glass slide with an approximately 25 z 25 array of mapped P1 clones, with each row of bound DNA strand being a series of five megabase resolution clones from a particular chromosome; higher resolution disease specific clone sets are also available. In GCGH, tumor tissue DNA is extracted and labeled with a red fluorophor while an equiwdent amount of control DNA is labeled with a green fluorophor. After mixing the DNA strands, denaturing, exposing them to the immobilized clones and washing away unbound strands, the ratio of captured red to green fluorophores is measured at each target area; red/green ratios of greater than and less than one indicate amplificatio~a and deletion, respectively. Miniaturization is critical for GCGH to become a practical diagnostic tool. For example, using a microscope based imaging system with sensitivity of 5 x 108 fluors/cm 2, a hybridization efficiency of 0.02% is suffi- cient for analysis if the spots are 100 /tin in diameter, however, hybridization efficiency must increase to 2% if a 1 mm diameter spot is ~ased.

Hybridization specificity can be increased by exploit- ing the inherent negative overall charge on DNA molecules to move and separate those molecules not hybridized to the immobilized probes; Michael Heller (Nanogen, San Diego, CA) presented proof of concept experiments for a chip based devise using this 'electronic stringency control' in which an addressable pro- gram electronic matrix (APEX) is employed to transport, bind, and separate charged molecules by electric fields. Prototype chips contained 5 x 5 and 8 x 8 arrays of addressable microlocations (each 80 /~m diameter, total chip dimensions ~ 1 cm2), each of which can be biased positive or negative via a platinum microelectrode. The biological test area is separated from the electrode by a permeation layer to isolate electrolysis products from the DNA solutions. A positive charge on the microelectrode attracts fluorescently labeled target DNA to immobilized probes to expedite hybridization. A reverse in the polarity results in removal of non-bound and mismatched template but does not affect those strands which are perfect comple- ments to immobilized probes. Both the speed and specificity of hybridization are enhanced using electronic stringency control; the target is concentrated at the microlocations within 4 s of initiation of the positive bias while conventional hybridization requires longer than 1 h. Using this technique, a perfectly matched Ras-12 oncogene target was differentiated from a target that contained a single base mismatch five times more effectively than by conventional hybridization.

Yu Hui Rogers (Molecular Tool, Baltimore, MD) discussed recent progress in the use of genetic bit analysis (GBA) for solid phase genotyping, which unlike pure hybridization based methods is not compro- mised by ambiguities due to probe base composition, temperature, buffer conditions, or duplex length. In GBA, a template containing a polymorphic region is annealed to an immobilized oligonucleotide in a fashion analogous to SBH. The immobilized primer is designed to terminate one base upstream of the variable site; following hybridization and the addition of a polymerase and four ddNTPs with different fluorescent labels, the identity of the base by which the primer is extended (and thus the template variable position) is revealed by the emission wavelength of its attached fluorophore. Thus while hybridization methods alone may not reliably discriminate single nucleotide poly- morphisms, GBA has increased specificity since the fidelity of DNA polymerases is quite high. Using fluorogenic detection substrates, GBA was employed to easily differentiate an A versus G polymorphism: for each, signal intensities from perfect matches were over an order of magnitude higher than those of the nucle- otides improperly incorporated. Heterozygotes are easily recognized since they result in contributions to the fluorescence spectrum from two dyes. Direct fluorescent

156 M.J. O'Donnell-Maloney, D.P. Little/Genetic Analysis: Biomolecular Engineering 13 (1996) 151-157

detection without enzymatic amplification, though quite attractive, is far more challenging since typical fluorescent detectors require on the order of 10 3 fluors/#m 2 surface, while only 102 fluors//zm 2 are generated in a typical GBA reaction. Initial experiments using silicon slides in place of the currently used glass slides showed improved sensitivity since the fluorescent background is considerably lower.

Elmer 9600, amplification efficiencies of the fl-actin gene were comparable to those from the larger, more expensive device. With the CCD camera, the emission spectra from each of the 24 wells on 1 chip could simultaneously be detected; thus the products of 96 reactions can be monitored in only four 'scans'.

3.3. Integrated chip systems

3.2. Miniaturized P C R devices

Advantages in speed, material consumed, and cost have catalyzed a swing in analytical and synthetic chemistry laboratories from macro- to micro-scale preparations; molecular biology on a reduced experimental scale also affords theoretical advantages to the field. The ubiquitous polymerase chain reaction is a logical starting point for miniaturization, and several groups presented such experimental data. Allen Northrup (Lawrence Livermore National Lab, Liver- more, CA) discussed the miniature analytical thermal cycler (MATCI), underscoring that the chemistry is the 'brain of diagnostics' and that the associated instrumentation should be small, inexpensive, and simple. The amplification and detection device is composed of centimeter-sized, silicon-based thermal chambers that are controlled by a hand-held battery operated con- troller consuming only 1.2 W per reaction chamber. The cycler is equipped with optical windows for real- time monitoring of product generation and can be interfaced to a microelectrophoresis (separation ~ 1 min) compartment after amplification ( ~ 20 min). Cy- cle times were reduced by an order of magnitude compared to conventional cyclers due to decreased thermal diffusion distances and more uniform thermal gradi- ents. While the amplification factor of cloned DNA is comparable to that of a conventional thermocycler, that of genomic DNA poses more problems; despite this disadvantage, multiplex amplification of seven mutant alleles of cystic fibrosis has been accomplished with MATCI.

Timothy Woudenberg (Perkin Elmer, Foster City, CA) also spoke of microscale PCR and the goal of integrating thermal cycling, fluorescent detection and application specific analysis into a single system. It is anticipated that this system could also combine real time detection of PCR growth curves using the Taqman fluorescent quenching assay. Additionally, after amplification, the PCR products could be loaded directly into either microcapillary electrophoresis or hybridization compartments. An integrated robotic station was demonstrated combining a PCR cycler with CCD visu- alization using four 12 x 12 mm chips (each with a separate temperature control) with 24 wells/chip. Al- though requiring only 2-10% of the template volume and 25% of the time per cycle compared to the Perkin

A major theme throughout the conference was the development of a means of providing methods for DNA diagnostics that require minimal human interven- tion (i.e. potential human error) between the steps. Such a mindset has spearheaded efforts toward integrated diagnostic/detection systems, with the holy grail being a 'push-button' instrument to go from blood to diagnostic product. In addition to simplification by interfacing the 'world-to-chip' (i.e. only add sample), the advantages afforded include decreased sample loss upon moving analytes to other devices and the lack of moving parts, thus decreasing manufacturing costs. The necessary process integration represents a significant experimental and engineering challenge. Both Michael Heller (Nanogen, San Diego, CA) and J. Wallace Parce (Caliper Technologies, Palo Alto, CA) outlined strate- gies for such integrated analysis chips which enable DNA extraction, appropriate reactions, separation of products and detection. One hurdle in the development of such a chip is moving the sample from one chamber to the next; transport of analyte or reagents via electric fields (electroosmotic pumping) is most suited for the Nanogen device, while Parce pointed out that acousti- cal pumping may also be suitable for other devices. In the proposed Nanogen prototype, the ( ~ 20/zl) biological analyte such as blood, for example, is injected into a cell selector which separates white from red blood cells; the selected cells are then transferred to a crude DNA selector where the genomic DNA is hybridized to probes specific to intron sequences such as Alu repeats. Following enzymatic digestion, fragments are transferred, again electronically, to a fragment selector com- ponent with approximately 200 addressable locations for specific hybridization of certain fragments to reduce the complexity of the mixture. After removal 6f un- wanted components, the fragments are delivered to the APEX previously described for multiplexed hybridization analysis. The fully integrated system can be contained on a chip less than 20 x 20 cm, and includes additional regions for DNA storage, reagent dispensing, and waste disposal. Similarly, Parce presented data from Mike Ramsey (Oak Ridge National Laboratory, Oak Ridge, TN) on initial experiments demonstrating an integrated device not containing the hybridization region. An integrated chip was employed for pBR322 digestion, electrophoretic separation of the fragments, and fluorescent detection; separation time of fragments ranging from 75-1632 bp was less than 3 min.

M.J. O'Donnell-Maloney, D.P. Little / Genetic Analysis: Biomolecular Engineering 13 (1996) 151-157 157

Acknowledgements

We gratefully acknowledge the support and helpful discussions provided by Dr. Hubert K6ster and Dr. Charles Cantor.

4. Conclusion

Recent years have seen dramatic advances in our ability to miniaturize existing 'macroscale' processes and microfabricate physical procedures. These ad-

vances are evidenced in basic array development, arrays used as diagnostic tools, as well as novel applications of arrays for sequencing by hybridization and related hybridization techniques. Not only have arrays evolved as of late, but numerous microma- chined devices are being developed to replace cumber- some conventional equipment and to integrate many procedures on the chip level. It is abundantly clear that molecular biologists are already gaining significant efficiency improvements by switching to miniaturized devices, and that the molecular biology laboratory of the 21st century will be considerably smaller and more cost effective.

microfabrication and array technologies for dna sequencing and diagnostics

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