|Genome Sequencing Technologies and Resources
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|15. Structural Analysis of the
T7 DNA Replication System and Further Development of its Use in DNA Sequencing
Stanley Tabor and Charles Richardson
DNA polymerases play an essential role in current methods of DNA sequencing, which require the efficient synthesis of DNA using the four natural nucleotides as well as analogs such as fluorescently-labeled nucleotides and chain-terminating dideoxynucleotides. We have been characterizing the structure and function of DNA polymerases in order to modify those properties that are important for DNA sequencing. Our work has focused on the DNA polymerases of the Pol I family, that includes T7 DNA polymerase and Taq DNA polymerase. We, in collaboration with Sylvie Doublié and Thomas Ellenberger, recently determined the 2.2 crystal structure of T7 DNA polymerase locked in a replicating complex with a dideoxy-terminated primer-template, an incoming dNTP, and the processivity factor thioredoxin1. We are using this structure to design and characterize mutations in the active site of T7 and Taq DNA polymerases that have altered specificity for analogs with modifications in the sugar moiety (e.g. dideoxynucleotides, ribonucleotides and 3' fluoro derivatives) and bases containing bulky fluorescent substituents.
One property that distinguishes T7 DNA polymerase from the thermophilic DNA polymerases used for DNA sequencing and amplification is its high processivity. This is achieved by the binding of its processivity factor, E. coli thioredoxin, to a unique 74 residue domain that acts as a flexible tether to keep the polymerase bound to DNA. While this domain is unique to T7 DNA polymerase, it is modular in that it can be transferred to other homologous polymerases by gene fusion to generate hybrid enzymes that have dramatically increased processivity2. The crystal structure of the T7 DNA polymerase complex suggests that thioredoxin is acting to stabilize the region it binds to, allowing a number of basic residues to interact electrostatically with the DNA backbone to prevent dissociation. We are characterizing mutations in this region in order to further define the critical structural features and to engineer new DNA polymerases that have increased processivity.
The complex of T7 DNA polymerase and T7 helicase/primase synthesize DNA with high efficiency. We have been optimizing reactions carried out by these enzymes in combination with other T7 replication proteins. Conditions have been developed in which DNA synthesis is exponential. Using one pg of plasmid DNA as template, a 15 min reaction can produce 10 g of product DNA, corresponding to a 10 million-fold amplification. DNA synthesis is nonspecific; the entire plasmid is replicated. We are exploring the use of this amplification reaction to produce BAC and plasmid DNA for use in DNA sequencing reactions. This in vitro synthesis of DNA may be an attractive alternative to the current methods that rely on in vivo production in bacterial cells for the automated preparation and purification of DNA templates.
This work is funded in part by DOE grant DE-FG02-96ER62251 (Stanley Tabor, P. I.)
1 Crystal Structure of Bacteriophage T7 DNA Polymerase Complexed to a Primer-Template, a Nucleoside Triphosphate, and its Processivity Factor Thioredoxin. Sylvie Doublié, Stanley Tabor, Alexander Long, Charles C. Richardson and Tom Ellenberger, Nature, 391, 251-258 (1998).
2 The Thioredoxin Binding Domain of Bacteriophage T7 DNA Polymerase Confers Processivity on Escherichia coli DNA Polymerase I. Ella Bedford, Stanley Tabor and Charles C. Richardson, Proc. Natl. Acad. Sci. USA 94, 479-484 (1997).
16. Mutagenesis and Reaction Condition Studies of T7 RNA Polymerase Variants to Incorporate Deoxynucleotides
Mark Knuth, Scott Lesley, Heath
Klock, Michelle Mandrekar, Ryan Olson, James Schaefer, and Kris Zimmerman
Our aim is to alter substrate specificity in T7 RNA polymerase for efficient incorporation of dNTPs and other nucleotide analogs. Such a promoter-directed polymerase could be used for DNA sequencing and creating hybridization probes without the need for initiating primer. Previously described mutants incorporate dNTPs, but not sufficiently for practical applications. We are undertaking a combined approach of site-directed mutagenesis and evaluating reaction conditions to create an efficient polymerase with altered nucleotide specificity. Results are shown for both efforts.
For mutagenesis, we superimpose all possible substitutions of individual target sites upon the previously described mutations. Approximately 200 sites, covering a large portion of the active site, were chosen for saturation mutagenesis. Evaluation is underway, and desirable substitutions will be combined and shuffled. Our previous results indicate that dNTP incorporation is inhibited by inefficient transition from the initiation to the elongation phase. In order to screen for improvement of this property, polymerase is purified from each mutant and a fluorescent assay used to determine its ability to incorporate mixtures of r/dNTPs.
Solution conditions, such as addition of organic solvents, have been reported to enhance dNTP incorporation but result in substantial reduction of activity. We have confirmed and extended these results and note that these agents lower the apparent denaturation temperature of T7 RNAP. Mutations which increase thermostability under these conditions might offset the activity decrease, and a screen for these is being incorporated in our mutagenesis approach. We also evaluated other agents and found several which also enhanced dNTP incorporation but with a lesser thermodestabilization. Our results suggest a conformational change in the protein rather than the DNA template may be responsible for this enhancement. Using our optimal conditions, incorporation of a mixture of 3 dNTPs / 1 rATP is 300% of the 4 rNTP value. Although transcript products are somewhat shorter (most <= 200 bp) than for 4 rNTPs, we are examining their performance in dye-terminator DNA sequencing.
16a. Full-length Mouse cDNA Analysis by Automated Fluorescent 384 Capillary Sequencer System (RISA; Riken Integrated Sequence Analysis System)
Hayashizaki Y, Okazaki Y, Kawai J, Carninci P, Shibata K, Ito
M, Tateno M, Sasaki N, Konno H, Sugahara Y, Kawahire S, Izawa M, Shibata
Y, Watahiki M, Yoneda Y, Tanaka T, Matuura S, Muramatsu, M
We have developed an automated fluorescent 384 capillary sequencer and a plasmid preparator that can extract a total of 40,000 plasmid clones in one day per a machine. A single automated sequencer can now process up to 2304 samples per day.
Full-length cDNA libraries based on the biotynlated cap trapper method have been developed and constructed. These full-length cDNA clones were picked into 384 well plates using Q-bot (Genetix inc.) and stored. We have sequenced more than 100,000 runs of 3'end sequences and classified them into more than 20,000 clusters. They are now open on the web (http://genome.rtc.riken.go.jp). We are now planning to sequence 1,000,000 runs in a coming year and categorizing the mouse full-length cDNAs.
17. Megabase and Gigabase Templates: Direct Automated Sequencing off Microbial and Eukaryotic Chromosomal DNA
Combination of the robust dye terminator and ThermoFidelase chemistries has provided solution for the automated sequencing directly from microbial Megabase-long templates. Novel approach streamlines gap closure in the large-scale projects and does not rely on extra subcloning or combinatorial PCR. Redesign of genome sequencing and gene hunting strategies promises to substantially reduce the volume of and eventually completely eliminate shotgun steps in microbial genome projects.
Our work indicates that Megabase sequencing chemistry provides results with high quality, sensitivity, reproducibility and speed. We will present data on the direct detection of single nucleotide polymorphisms (SNPs) and gross sequence variations between genomic regions from a number of closely and distantly related microorganisms. We will discuss technical and economic feasibility of the new strategy in large-scale projects on comparative genomics.
The development of sequencing chemistry for Gigabase templates such as human and other complex eukaryotic genomes is one of the most challenging tasks in technology development. Our initial work is focused on the specifics of preparation and handling of Gigabase templates, target selection, achievement of sufficient fluorescent signal strength and quality. We will review basic techniques used in the development of novel chemistry and present our first successful results on the automated sequencing directly from fish and human chromosomal DNA.
This work is supported in part by DOE grant DE-FG02-98ER82557 and NIH grant 2R44GM55485-02.
18. PCR Using Branched Modular Primers
Maura M. Devine, Mugasimangalam C. Raja,
and Levy E. Ulanovsky
Here we present a novel PCR technique termed "branched primer PCR" which eliminates the need for custom primer synthesis by combining oligonucleotide modules selected from a pre-synthesized library. A branched primer involves two oligonucleotide modules that are physically linked by annealing to each other as well as to the target, forming a three-way junction. Branched primers were developed (initially for DNA sequencing rather than for PCR) as a type of modular primer whose modules anneal cooperatively to the template. This cooperativity is provided by mutually complementary segments in the two modules that bind to each other forming what is termed a "stem" region. Before actual PCR can take place, a branched primer is extended along the template. This extension strand is then used as the template for a reverse branched primer extension. The reverse extension product is then amplified using PCR primers homologous to the stems of each branched primer. These PCR primers are universal in that the stem sequence is the same in different branched primers. In contrast, the sequences of the stretches which are complementary to the template are variable throughout the presynthesized library of the oligonucleotide modules (each in a separate tube). Additional sequence-specificity of PCR is provided by nesting. Branched primer PCR is expected to be useful for applications such as resequencing closely related genomes (e.g. rodents and primates) which require a huge number of custom PCR primers. The latter would then be conveniently replaced with a much smaller library of presynthesized oligonucleotide modules for branched primers (2,000 to 4,000 modules instead of millions of custom PCR primers).
19. Synthesis, Characterization, and Potential Applications of Biotinylated Energy Transfer Oligonucleotides
Jin Xie1, Richard A.
Mathies2, and Alexander N. Glazer1
Energy transfer (ET) fluorescent dye-labeled primers have provided a decadic improvement in the performance of DNA sequencers for high-throughput sequencing1,2. The acceptor emissions of high spectral purity also make ET primers ideal for diagnostic applications, such as forensic identification and genetic typing of short tandem repeats3. Biotin has an extraordinarily high affinity for streptavidin with a reported dissociation constant of ~10-15. This very strong binding affinity has made the biotin-streptavidin system very attractive for a multitude of in vitro labeling applications. We describe here the synthesis and characterization of biotinylated fluorescent ET reagents. Hung et al. have shown that CYA-ROX primers with a donor-acceptor spacing of 8-10 nucleotides offer excellent acceptor emission intensities coupled with negligible donor emissions4-7. We have synthesized oligonucleotides with the sequence 5'-CYA- NNNNNNNNNTROXNNTBNNNNNNN-3' with donor-acceptor fluorophore pairs separated by 10 intervening nucleobases, but varying in the location of TB, in this example introduced two bases 3' to the base carrying the acceptor ROX. Biotin-labeled T (TB) was introduced by the use of biotin-dT phosphoramidite at different locations in the oligonucleotides. CYA, 3-(e-carboxypentyl)- 3'-ethyl-5,5'-dimethyloxacarbo-cyanine, a dye with a high absorption cross-section but a low fluorescence quantum yield, was chosen as an energy donor at the 5'-end of the oligonucleotides, and ROX as an acceptor was attached to a modified thymidine (TROX). We have compared the quantitative spectroscopic properties of four biotinylated ET reagents differing in the spacing between donor-biotin pairs and acceptor-biotin pairs. CYA10ROX- 2-Biotin (where 2 is the number of nucleotides between the acceptor and biotin) reagent offers the best combination of acceptor fluorescence emission intensity and spectral purity. With 488-nm excitation, the fluorescence emission intensity of C10R-2-Biotin is 16-fold stronger than that of the corresponding oligonucleotide labeled with the acceptor ROX as the only dye. These biotinylated ET reagents have a broad range of potential applications, e.g., affinity purification and detection in DNA mapping applications on chips, and in cell sorting8. For such purposes, we have prepared and characterized ET-oligonucleotide-streptavidin conjugates for use in multiplexed assay systems.
1J. Ju, A.N. Glazer, and R.A. Mathies Nature Medicine 2, 246-249 (1996).
2A.N. Glazer and R.A. Mathies Curr. Opinion Biotechnol. 8, 94-102 (1997).
3Y. Wang, S-C. Hung, J.F. Linn, G. Steiner, A.N. Glazer, D. Sidransky, and R.A. Mathies Electrophoresis 18, 1742-1749 (1997).
4S-C. Hung, J. Ju, R.A. Mathies, and A.N. Glazer Anal. Biochem. 243, 15-27 (1997).
5S-C. Hung, J. Ju, R.A. Mathies, and A.N. Glazer Anal. Biochem. 238, 165-170 (1996)
6S-C. Hung, R.A. Mathies, and A.N. Glazer Anal. Biochem. 252, 78-88 (1997).
7S-C. Hung, R.A. Mathies, and A.N. Glazer Anal. Biochem. 255, 32-38 (1998).
8See abstract "Integrated Sequencing Sample Preparation on CE Microplate" by Y. Shi, I. Kheterpal, J. Xie, A.N. Glazer and R.A. Mathies.
20. Development of a Multilabel DNA Mapping Technique Using SERS Gene Probes
Tuan Vo-Dinh1, David
L. Stokes1, Guy D. Griffin1, Jean-Pierre Alarie1,
Edward J. Michaud1, Terry Bunde1, Ung-Jin Kim2,
Melvin I. Simon2
We report the development of a novel approach for use in DNA mapping and bacterial artificial chromosomes (BAC) colony hybridization using a unique type of DNA gene probe based on surface-enhanced Raman scattering (SERS) labels. An important step toward sequencing the human genome involves assembling ordered, overlapping sets (contigs) of clones that have been mapped and well characterized. A unique approach that would greatly facilitate large-scale genomic sequencing involves building genome-wide BAC-based contig maps.
In this work, we have developed various types of SERS-active substrates that can be used to provide this "label-multiplex" capability, thereby reducing the time for genome characterization. We have developed various schemes for binding SERS labels onto DNA targets for use in BAC clone mapping. We have demonstrated the feasibility of a multi-label SERS detection scheme, whereby multiple labels can be detected simultaneously in each multiplex probing cycle. Raman spectroscopy is an important analytical tool due to its excellent specificity for chemical group identification. With the use of the SERS effect, Raman scattering efficiency can be enhanced by factors of up to 108 when a compound is adsorbed on or near special metal substrates1. The surface-enhanced Raman gene (SERGen) probes do not require the use of radioactive labels and have a great potential to provide both sensitivity and selectivity for DNA mapping and sequencing. The method is aimed at simultaneous detection of multiple probes for DNA mapping/sequencing using BAC clone applications. The technology is designed to be versatile and broad-based, and to allow a rapid and shortcut approach to significantly improve the speed of BAC colony hybridization. cDNAs are an excellent resource to rapidly build genome wide BAC contigs. They represent inexpensive, efficient probes to screen BAC libraries by colony hybridization. However, the conventional approach relying on 32P-labeled probes is laborious and time consuming. Multiplexing probes with non-radiative chemicals that can be efficiently distinguished after hybridization will greatly reduce the time and effort for establishing the large-scale BAC library that is required for characterizing the entire genome of human, mouse and other organisms.
This research is sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy under contract DE AC05-960R22464 with Lockheed Martin Energy Research Corporation.
1T. Vo-Dinh, "Surface-enhanced Raman spectroscopy using metallic nanostructures" in Trends in Analytical Chemistry, 17, 557 (1998)
20a. Construction of a Genome-Wide, Highly Characterized Clone Resource for Genome Sequencing
Gregory G. Mahairas, Keith D. Zackrone, Stephanie Tipton, Sarah Schmidt,
Alan Blanchard, Anne West, and Leroy Hood
As the genome project shifts into the large-scale sequencing phase, an overwhelming technical challenge resides in developing an efficient method for producing minimum tiling paths of sequence-ready clones across the entire genome. BACs are becoming the large fragment clones of choice among sequencing centers and BAC clones provide significant advantages as source material for sequencing (Kim et al., 1996a). BAC clones are stable, have a small vector size (7.4 kb), can be sequenced directly by the shotgun approach, are sufficiently long to traverse most tandem arrays of homology units or genome-wide repeats, and have been shown to be randomly distributed across the human genome (Kim et al., 1996b). By the fall of 1998, together with The Institute for Genome Research (TIGR), we will have sequenced the BAC ends or sequence tagged connectors (STCs) from 150,000 clones (e.g. 300,000 STCs --150,000 from each laboratory). We have also generated a HinDIII restriction digest for each BAC whose end sequences have been determined at the University of Washington. We have developed a strategy and tools for using this resource in support of large-scale genomic sequencing and demonstrated proof of concept for its use. Together with TIGR, we propose to complete the characterization of an STC clone resource from two IRB-approved human BAC libraries to 22.5-fold clone (BAC) coverage (e.g. 450,000 BAC clones assuming an average insert size of 150 kb). These data will be immediately available on the world wide web through dbGSS and our web sites (www.genome.washington.edu and www.tigr.org) and the clones will be available for distribution to the scientific community through Research Genetics. Nine hundred thousand STC sequences will provide a sequence marker of 300 to 500 base pairs (bp) on average every 3,100 bp across the genome. The BAC libraries and the data pertaining to them will enable the facile selection of minimum tiling paths of BAC clones across each of the human chromosomes for large-scale sequence analysis (see below).
1) By fall of 1998, TIGR and the University of Washington High-Throughput Sequence Center (HTSC) will have each sequenced 150,000 STCs (total 300,000). By fall of 1999, TIGR and the University of Washington HTSC each propose to sequence an additional 300,000 STCs for a total of 900,000 STCs. This will, on average, place 1 STC every 3.1 kb across the genome and provide a 22.5-fold clone coverage of the genome. All clones sequenced after June of 1998 will come from IRB-approved BAC libraries.
2) Produce a restriction map of each BAC clone characterized at the University of Washington. These will be useful for identifying clones that contain no inserts or short inserts for verification of genomic fidelity and for checking the assembly process.
3) Continue the development of tools to extract biologically relevant information from the data and utilize the resource for high-throughput genomic sequencing.
21. Vectors for Using Nested Deletions to Sequence Either Strand of Cloned DNA
John J. Dunn, Laura Praissman, Laura-Li
Butler-Loffredo, John J. McNulty, and F. William Studier
Regions of highly repeated DNA are encountered frequently in human DNA and are likely to be particularly troublesome near centromeres and telomeres. Highly repeated regions are difficult to assemble correctly by shotgun sequencing, but cloned fragments at least 10 kilobase pairs long can be sequenced and assembled easily by generating an ordered set of nested deletions whose ends are separated by less than the length of sequence read from a single priming site within the adjacent vector. Assembly of the overlapping sequences is guided by knowledge of the relative length of the portion of the fragment remaining in the clone, as determined by gel electrophoresis.
We have made a series of plasmid vectors, the pZIP series, which allow rapid generation of an ordered set of nested deletions from either strand of a cloned DNA fragment. The vectors are based on the low-copy F replicon. The size of the vector DNA has been reduced to the 4.5-kbp range by removing the 2.5-kbp sop (stability of plasmid genes) region. The resulting plasmids have the low copy number typical of F plasmids and remain stable enough to be easily maintained by growth in the presence of kanamycin, the selective antibiotic. DNA in amounts convenient for sequencing is readily obtained by amplification from an IPTG-inducible P1 lytic replicon.
Nested deletions are generated by cleavage near one end of the cloned fragment, using commercially available site-specific endonucleases (PI-PspI, I-CeuI or I-SceI) whose recognition sites span 18-30 bp and are therefore unlikely to occur in cloned DNA fragments. Cleavage by these nucleases generates four-base 3' overhangs that are resistant to digestion by E. coli exonuclease III. A second cleavage by one of several nucleases with 8-base recognition sites leaves the end adjacent to the cloned fragment susceptible to ExoIII digestion, permitting unidirectional 3' to 5' digestion across the cloned fragment. The resulting single-strand tails are digested with S1 nuclease, the ends are repaired and ligated with T4 DNA polymerase and ligase, and clones are obtained by electroporation. An range of digestion times with ExoIII can easily produce a distribution of deletion lengths extending across the entire cloned fragment. Cleavage sites for the site-specific endonucleases are positioned in the vectors so that nested deletions can be generated from either end of an individual cloned fragment.
Conditions for routinely generating ordered sets of nested deletions and using them to sequence both strands of cloned fragments in the 5-kbp to 15-kbp size range are being developed by sequencing fragments of human DNA from BACs.
22. Direct Conversion of PCR Products into Bidirectional Sequencing Fragments
Kenneth W. Porter, Ahmad Hasan, Kaizhang
He, Jack Summers, and Barbara Ramsay Shaw
The search for more efficient and direct approaches to genomic sequencing continues to gain attention, particularly in gap-filling, finishing, and diagnostic applications. We have developed an alternate sequencing chemistry which avoids cycle sequencing, allows direct bidirectional genomic sequencing, and permits direct loading of PCR products onto the separating system. The method employs template-directed enzymatic, random incorporation of small amounts of boron-modified nucleotides (i.e. 2'-deoxynucleoside 5'-alpha-[P-borano]- triphosphates) during PCR amplification. The position of the modified nucleotide in each PCR product can be revealed in two ways, either enzymatically (as previously described1) or chemically. Both approaches take advantage of differences in reactivity of the normal and modified nucleotidic linkages to generate PCR sequencing fragments that terminate at the site of incorporation of the modified nucleotide. By employing labeled PCR primers, the original PCR products are able to be converted directly into bidirectional sequencing fragments.
In the enzymatic approach, the modification of a phosphate into a boranophosphate internucleotidic linkage prolongs its lifetime toward degradation by nucleases. The sequential hydrolysis by 3'-5' exonuclease III is thereby blocked by a boranophosphate, resulting in fragments that terminate in a boranophosphate nucleoside. However, normal and boranophosphate linkages with a 3'-cytosine are more susceptible to exonuclease degradation than other purines and pyrimidines, which reduces band uniformity. A series of base-modified cytosine derivatives were therefore synthesized and tested for nuclease resistance. The 5-ethyl-alpha-borano-dCTP analog was found to exhibit an increased resistance to exonuclease III compared to the alpha-borano-dCTP used previously in our method, without affecting incorporation, and resulted in more even banding patterns. Analysis with Basefinder software (M. Giddings) takes into account any mobility changes, permitting increased consistency and accuracy. The enzymatic approach may find use in applications where high resolution of longer fragments requires stronger signals at longer read lengths, because the distribution of fragments produced by nuclease digestion is skewed to long fragments.
We are also developing a chemical method for generating sequencing fragments, as an alternative to exonuclease chew-back. In the chemical approach, we have identified reagents that selectively cleave the backbone of the PCR product at boranophosphate linkages, while leaving the normal phosphodiester linkages intact. We anticipate that chemical cleavage following incorporation of fluorescently labeled borano-dNTPs may result in a more efficient method of sequencing. Also under investigation are agents that can result in colorimetric detection of boranophosphate.
Direct sequencing of PCR products simplifies mono- and bidirectional sequencing and provides a simple, direct, and complementary method to cycle sequencing.
1K.W. Porter, J. D. Briley, and B. R. Shaw, "One-Step PCR Sequencing with Boronated Nucleotides", Nucleic Acids Research 25, 1611-1617 (1997).
23. Analysis of Gradients of Polymer Concentration or Ionic Strength
Mark A. Quesada, David J. Fisk,
and F. William Studier
We are investigating whether gradients of polymer concentration or ionic strength can extend read lengths into the 1000-2000 base range when analyzing DNA sequencing reactions by capillary electrophoresis. Longer read lengths would increase sequencing efficiency and reduce the effort needed in the assembly and finishing stages of genome sequencing.
Gradients of polymer concentration or ionic strength along the length of the capillary are generated by merging two solutions in the capillary, using programmable syringe pumps. Parameters important for obtaining reproducible gradients were identified and controlled with the aid of fluorescent dye to analyze the distribution of one of the solutions along the length of the capillary. Because entangled polymer solutions have high viscosity, balancing hydrostatic conductance at the junction between the merging solutions is critical for producing well defined, reproducible gradients. A smooth gradient with uniform composition in the radial direction at each capillary cross section is established by radial diffusion, primarily of water and other low molecular weight components within the capillary (polymer swelling). Production of reproducible gradients requires merging the solutions in a controlled way at a sufficiently low flow rate, and allowing sufficient time for diffusion to create a smooth and uniform gradient before the capillary is used for analysis.
Once conditions were established for preparing reproducible and useful gradients of polymer concentration in capillaries, the effects of different gradient configurations on read length were examined. We expected that a gradient of increasing polymer concentration could counter the peak-broadening effects that are responsible for loss of resolution at long read lengths, and this appears to be the case. Certain gradient configurations yield single-base resolution near 800 bases in standard, room-temperature analyses with separations continuing well beyond 1000 bases. Gradients of increasing ionic strength (salt concentration) might be expected to have a similar band sharpening effect, and we are beginning to explore resolution in different combinations of polymer and salt gradients.
If the band sharpening effects of polymer or salt gradients can increase resolution and extend DNA read lengths in capillary electrophoresis, the same concepts may also find application for improving the performance of microchannel systems and disposable chips.
24. Design and Assembly of a Turnkey, High Throughput Oligonucleotide Synthesis Facility for Use on the Human Genome Project
J. Shawn Roach and Harold R. Garner
The objective of this project was to design and assemble a highly automated, high throughput oligonucleotide synthesis facility that requires as little operator intervention as is practical. Each of the pre-processing and post-processing steps required for high throughput oligosynthesis were examined for opportunities to streamline and automate if practical. These steps include 1) loading the solid supports into the filter plates prior to synthesis, 2) cleaving the oligos from the solid supports post synthesis, 3) rapid evaporation of the residual solutions after chemical deprotection, 4) optical density evaluation of the oligo concentration after resuspension, 5) gel electrophoresis evaluation of the oligo quality, and 6) automated dilution of the oligos to a normalized concentration after resuspension.
The heart of the system is two MerMade high throughput oligonucleotide synthesizers that are each capable of synthesizing up to 192 oligos a day. The oligos are synthesized on solid supports loaded into two 96-well filter plates. Standard phosphoramidite chemistry is used to perform the synthesis. The MerMade was designed by Dr. Harold R. Garner and Dr. Simon Rayner of the University of Texas Southwestern Medical Center. The MerMades used on this project were built by Avantech Automation Corporation (New Braunfels, TX) according to the Garner-Rayner design with minor modifications. Additional devices in the facility include a Biomek 2000 Workstation (Beckman Instruments, Fullerton, CA) for performing several of the liquid transfer and filtration steps necessary, a Jetstream evaporator and Scirocco gas heater system (Helix Scientific, Warminster, PA) for rapid sample concentration, and a Spectramax 190 Plus UV-Vis spectrophotometer and 96-well plate reader (Molecular Devices, Sunnyvale, CA) for optical density analysis.
Data from oligonucleotides synthesized by the facility will be presented along with background information on the turnkey synthesis facility.
25. Prep Track I - A Dynamic Approach to Liquid Handing Robotics
D. Humphries, M. Pollard, J. Bercovitz,
C. Reiter, and B. Gray
Prep Track I is a modular liquid handling robot that was developed at LBNL for the Human Genome Project. This machine has been in productive operation for more than a year. The basic function of this machine is to process solutions in micro-titer plates that are removed from input cassettes and fed to two conveyor belts where various liquid processes are performed by 96 syringe multi-dispensers or hydra heads. After processing, the plates are automatically loaded into output cassettes. The hydra heads of each module have access to automated wash and rinse baths and, for some modules, a cold plate and rinse bath system. Production activity on Prep Track I has indicated a need for increased capability and flexibility in the physical infrastructure of the device. Towards this end, innovations from the newer Prep Track II are being applied to Prep Track I to allow a flexible three bath system with an interchangeable cold plate arrangement. A programmable valve manifold and multiple reservoirs will allow rapid reconfiguration of the bath/supply system by simply changing protocols and in some cases switching the physical order of baths and cold plates. The detail design process for Prep Track I is in turn feeding forward into Prep Track II to increase its capability and flexibility.
26. PrepTrack II Design: Lessons Learned from PrepTrack I
John Bercovitz, Martin Pollard,
David Humphries, Mario Cepeda, Charlie Reiter, and Bruce Gray
The PrepTrack is an automated high-throughput microtiter plate liquid handling machine. It is a linear modular system in which microtiter plates are transported from module to module by means of a conveyor belt. The instrument is designed to make automated efficient use of the Robbins Scientific pipetting head (96 or 384 syringes). All of the modules can operate simultaneously to perform liquid handling operations on individual microtiter plates or liquid transfers between microtiter plates. Each pipetting module is equipped with automated self-cleaning water and bleach baths. Efficiencies are realized by the parallelism of the Robbins pipetting head, the parallelism of multiple modules operating simultaneously, and the ability to continue pipetting operations on available modules while other modules are occupied with self-cleaning procedures.
The first PrepTrack machine has been in production use for one year. We have since completed a second PrepTrack machine. There were several design changes based on lessons learned from use of PrepTrack I. The design changes and reasoning behind the new designs will be discussed.
27. Adapting the Tecan Genesis 2 Meter Workstation for High Density Agarose Gel Loading
Linda Sindelar, John Bercovitz,
Mario Cepeda, and David Humphries
We have modified the Tecan Genesis Workstation to load high density agarose gels that are used for transposon mapping. Each Gel contains 210 wells that are 1mm wide and have a center to center spacing of 2.25mm. To achieve the positional accuracy required for this application we have designed modified pipetting tips and elevated work decks that hold four gels, source microtiter plates, markers, and a custom tip calibration fixture. We have also designed and fabricated casting trays and combs which precisely locate the gels on the work deck. Four gels are loaded with buffer and sample in 32 minutes. The application was written in the Tecan Logic Software.
28. Technology Development for the Human Genome Project
Chris Robinson1, Todd Brooks1,
Travis Crane1, Chris Elkin2 and Trevor L. Hawkins1,2
We are continuing our work on integrated robotic systems and technology development to aid the genome program. The integrated robotic approaches follow on from our development of the Sequatron robotic systems some years ago. Now, we are focusing on reducing volumes of the amplification and sequencing reactions as well as using higher density plates to perform these reactions. This all requires the development of new or modified hardware, such as thermal cyclers, which once developed will be modules for a new fully integrated system.
We are also working on improvements to the existing bottlenecks in high throughput DNA sequencing. One has been the development of a very low cost adaptation to the ABI 377 system that allows 96 lane gels to be run with the same results as found with the commercially available upgrade. Another, is the automated loading and pre running of ABI 377 gels for use in a high throughput facility.
Lastly, we are exploring the use of MALDI Mass spectrometry as a tool for the analysis of DNA extension products, specifically the resolution of compressions and error detection in genomic sequencing projects.
29. Automation for High Throughput Genomic DNA Sequencing
Ronald W. Davis
Stanford has developed several devices
that can be used as elements in a high throughput sequencing environment.
Among the instruments in development are thermal cycler and plasmid purification
devices. Stanford is working with the Joint Genome Institute on the incorporation
of selected instruments into their production environment.
The Joint Genome Institute (JGI) and Whitehead Institute will establish a Co-Development Program to produce an automated DNA sequencing production line with a capacity of 200 Mb per year. The production line will consist of:
31. Laboratory Automation for Finish Sequencing at LLNL
Stephan Trong, Arthur Kobayashi,
David J. Ow, Matt P. Nolan, Tom Slezak, Stephanie A. Stilwagen, Glenda
G. Quan, and Jane Lamerdin
The Human Genome Center at LLNL is performing high throughput DNA sequencing of the human genome. In the past year, we have contributed over 8.6 MB of high-quality finished sequencing to the Joint Genome Institute's total of 20.9 MB. This ramp represents an increase of more than 500% over the amount finished by LLNL last year (1.5 MB). One of the contributing factors in achieving this goal was the automation of sample processing through the use of robotic workstations.
In the finishing phase, we are currently processing an average of 9,000 samples per month with expectations of a 25-50% increase in the coming year. To meet this high volume of sample processing, we have employed the use of Tecan Genesis 150 liquid handling robots to rearray DNA templates into 96-well plates and for setting up sequencing reactions for the rearrayed clones/templates. To integrate this process with our sample-tracking system, we have developed a web-based system to perform the following functions:
This work was performed by Lawrence Livermore National Laboratory under the auspices of the U.S. Department of Energy, Contract No. W-7405-Eng-48.
32. Sheath-Flow Capillary Array DNA Sequencer Development at JGI/LBNL
Jian Jin, William F. Kolbe, Yunian
Lou, Earl W. Cornell, Alex Cheung, and Joseph M. Jaklevic
We have developed a 96-channel capillary electrophoresis system capable of production-level sequencing at increased rates and, more importantly, with improved automation. The system is based on an adaptation of the best available technology developed by several laboratories. In particular, we employ the sheath-flow excitation/detection geometry and a DNA sequencing protocol using linear polyacrylamide as sieving media. In addition, we have developed an effective base-calling software platform using a combination of algorithms. The sequencing system is fully integrated and includes a fixture for off-line capillary-array coating, gel-replacement and sample loading. The four-color sequencing instrument employs a cooled-CCD camera for data acquisition. Our custom base-calling software has been fully integrated with existing assembly algorithms including calibrated Phred scores and Phrap-based assembly. Currently we achieve a total run time of less than two hours, separating 750 bases per channel, with an average read-length (defined as having a Phred score > 20) of 350-400 bases/trace. The turn-around time between runs is less than 5 minutes. Over the past 9 months we have conducted a full evaluation of the system using sequencing templates taken directly from JGI production runs. During this time we generated more than 6 Mb of raw sequence data for system performance evaluation and protocol development. Those results have demonstrated that our system produces sequencing data with a quality comparable to commercial slab-gel systems currently employed in production sequencing. Detailed comparison data will be presented and future plans and discussed.
This work was supported by the Director, Office of Energy Research, Human Genome Program, of the U.S. Department of Energy under Contract N0.DE-AC03-76SF00098
33. Fully Automated DNA Sequencing with a Commercial 96-Capillary Array Instrument
Qingbo Li, Thomas E. Kane, Changsheng
Liu, Harry Zhao, Robert Fields, and John Kernan
A commercial high-throughput DNA sequencer has been developed based on a robust multiplexed 96-capillary electrophoresis system and a high-performance replaceable gel matrix. The instrument is fully automated with all the operation steps carried out and controlled by the instrument computer. The detector system employs on-column laser-induced fluorescence detection with an air-cooled argon ion laser as the excitation source. The instrument allows automatic processing of many 96-well sample trays without human intervention.
Including the time for sample introduction, separation, and capillary reconditioning, the instrument is capable of one complete run within two hours. The sequencing throughput of half million bases per day is readily achievable using this 96-capillary instrument. The instrument is also compatible with other DNA fragment analysis. With the experience of successfully building the 96-capillary instrument, the team is taking a further step toward developing a 384-capillary instrument.
Testing results will be presented for a fully functional 96-capillary instrument. Performance data of the instrument will be discussed.
34. Automation and Integration of Multiplexed On-Line Sample Preparation with Capillary Electrophoresis for High-Throughput DNA Sequencing
Edward S. Yeung, Hongdong Tan, and
An integrated and multiplexed on-line instrument starting from DNA templates to their primary sequences has been demonstrated based on multiplexed microfluidics and capillary array electrophoresis. The instrument automatically processes 8 templates through reaction, purification, denaturation, preconcentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were utilized to manage flow and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot-air thermal cycler. Subsequently, the sequencing ladders are directly loaded into separate size exclusion chromatographic columns operated at ~60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at ~70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. The raw data allow base calling up to 460 bp with an accuracy of 98%. The system is scalable to a 96-capillary array and will benefit not only high-speed, high-throughput DNA sequencing but also genetic typing.
An automated and integrated system for DNA typing directly from blood samples has been developed. The multiplexed eight-array system is based on capillary microfluidics and capillary array electrophoresis. Three short-tandem-repeat loci, vWA, THO1 and TPOX, are co-amplified simultaneously in a fused-silica capillary by a hot-air thermocycler. Blood is directly used as the template for polymerase chain reaction. Modifications of standard protocols are necessary for direct PCR from blood. A programmable syringe pump plus a set of multiplexed liquid nitrogen freeze/thaw switching valves are employed for liquid handling in the fluid distribution network. The system fully integrates sample loading, PCR, addition of an absolute standard, on-line injection of sample and standards, separation and detection. The genotypes from blood samples can be clearly identified in eight parallel channels when the electropherograms are compared with that of the standard allelic ladder by itself. Regeneration and cleaning of the entire system prior to subsequent runs are also integrated into the instrument. The system can be expanded to hundreds of capillaries to achieve even higher throughput.
35. Long-Read DNA Sequencing by Capillary Array Electrophoresis
Oscar Salas-Solano, Lev Kotler, Zoran Sosic,
Arthur W. Miller, Yongwu Yang, Haihong Zhou, and Barry L. Karger
The increasing prominence of capillary array electrophoresis for DNA sequencing raises the importance of being able to obtain long reads on a regular basis with such instruments. We have recently reported the use of capillary electrophoresis (CE) for routine DNA sequencing of 1000 bases in less than one hour using replaceable linear polyacrylamide solutions (Salas-Solano et al., Anal. Chem. 1998, 70, 3996-4003). These results have now been extended to a multiple-capillary array. Cycle-sequencing reactions, and most steps of subsequent sample purification, are performed in 96-well microtiter plates with a system built around a Biomek 2000 robot. Before each run, the array of polyvinyl alcohol-coated capillaries is refilled with linear polyacrylamide solution. The 488 nm output of an argon laser is directed into an optic fiber, and then into a line generator, which focuses the beam into a 35-micrometer wide line extending across the entire bank of capillaries. The emitted dye fluorescence passes through notch filters, and through a transmission grating for spectral dispersion, and is imaged onto a CCD that is read at 3 Hz. This design has no moving parts and is easy to align. Base-calling is done by an expert system (see separate abstract of A. W. Miller and B. L. Karger), and read lengths of 1000 bases and above at 98-99% accuracy are routinely obtained. We will also report on our latest results for achieving long read length sequencing using capillary electrophoresis with replaceable polymer solutions.
This work is being supported by DOE grant DE-FG02-98ER 69895.
36. DNA Sequencing Using Capillary Array Electrophoresis
Indu Kheterpal1, Gary
T. Wedemeyer1, Yuping Cai2, Alexander N. Glazer2,
and Richard A. Mathies1
Capillary array electrophoresis has emerged as a valuable tool for DNA analysis. We are developing methods for obtaining high quality sequencing separations using confocal fluorescence CAE instruments1,2 and energy transfer (ET) primers3. In practice replaceable separation matrices and base calling programs have been evaluated and optimized for high throughput sequencing separations of genomic DNA fragments from the cyanobacterium Anabaena.
We have evaluated the available replaceable gels using the same sequencing samples, temperature, detection system, injection and separation conditions. These gels can be pumped into the capillaries allowing the use of capillaries for potentially 100 runs. The three gels evaluated were linear polyacrylamide (LPA), hydroxyethylcellulose (HEC) and a mixture of HEC and polyethylene oxide (PEO). We have found LPA to provide the best sequencing separations with the longest read lengths of 1000 bases in the least amount of time. We have also evaluated several base calling packages for their ease-of-use, ability to batch process and base-calling performance. BaseFinder 4 has emerged as the leading program for our data and is being used for all of the sequencing data analysis.
We are currently validating methods by incorporating them into our Anabaena sequencing project performed by undergraduates. Several 6-15 kbp libraries of Anabaena genome potentially involved in the biosynthesis and control of phycobiliproteins have been constructed. The templates for sequencing are prepared by partial digestion of the genomic DNA utilizing restriction enzymes cocktails. Fragments in ~0.5 kb range are cloned into pUC 19 for bidirectional sequencing. The sequencing samples are generated using the Sanger dideoxy method and cycle sequencing. The separations are performed using our planar CAE instruments2 and replaceable gels. The data are analyzed using BaseFinder and assembled using Phred, Phrap and Consed. Two libraries p69 and p74 have now been completely assembled and sequencing on five other libraries is near completion. These libraries total over 70,000 bases and the fragments are being sequenced with ~5-fold redundancy to ensure complete and accurate assembly.
1 Huang, X. C.; Mathies, R. A. (1992) Nature (London), 359, 167-169.
2 Kheterpal, I.; Scherer, J. R.; Clark, S. M.; Radhakrishnan, A; Ju, J.; Ginther, C. L.; Sensabaugh, G. F. and Mathies, R. A. (1996) Electrophoresis, 17, 1852-1859.
3 Ju, J.; Glazer, A. N.; Mathies, R. A. (1996) Nature Medicine, 2, 246-249.
4 Giddings, M. C.; Severin, J; Westphall, M; Wu, J. Z; and Smith, L. M. (1998) Genome Research 8, 644-665.
37. Focused Single Molecule DNA Detection in Microfabricated Capillary Electrophoresis Chips
Brian B. Haab and Richard A. Mathies
Single-molecule fluorescence burst counting is a highly sensitive method for detecting electrophoretic separations of ds-DNA fragments1 with applications in environmental monitoring and health care diagnostics. We previously presented methods for optimizing dye labeling, laser power and data analysis, and conventional CE separations of ds DNA fragments in the 100-1000 bp range were detectable when only 50-100 molecules passed through the probe volume.2 We have now performed single DNA molecule detection in glass capillary electrophoresis (CE) chips which offer improved optics, faster separations, and increased molecular detection efficiency compared to conventional capillaries.3 Chips were fabricated with a 145 mm thick top plate that was matched to the design specifications of the 100X, 1.3 NA objective, yielding a two-fold increase in light collection efficiency. The channels were designed to focus a greater number of molecules through the laser beam to achieve enhanced detection sensitivity. The sample was constricted in the region of the 1 mm diameter focused laser beam by physical narrowing of the separation channel and by electrokinetic focusing caused by additional side channels in the detection region. The sample stream width decreased and the single molecule count rate increased linearly with the focusing current density. A four-fold improvement in molecular detection efficiency was achieved while maintaining single molecule sensitivity. The CE separation of a 500 bp PCR product was then detected using molecular focusing, which showed a two-fold increase in signal compared with conventional detection. A 300 fM sample was easily detectable with a signal-to-noise ratio of eight. These developments will enhance our ability to use CE separations to detect trace pathogen contamination or DNA mutation.
1 B. B. Haab and R. A. Mathies, Anal. Chem. 34, 3253-3260 (1995)
2 B. B. Haab and R. A. Mathies, Appl. Spec. 51, 1579-1584 (1997)
3 B. B. Haab and R. A. Mathies, Proc. SPIE 3259, 104-112 (1998)
38. Ultra-High Throughput DNA Genotyping and Sequencing on Radial Capillary Array Electrophoresis Microplates
Peter C. Simpson, James R. Scherer,
Yining Shi, and Richard A. Mathies
The microfabrication of DNA sample preparation, electrophoretic separation and detection devices is making possible a new generation of high-speed, high-throughput DNA analysis systems. Our research is focused on the ultra-high throughput analysis of PCR products for genotyping applications as well as DNA sequencing on microfabricated capillary array electrophoresis (CAE) microplates. These CAE microplates perform high speed analysis of multiple samples in parallel increasing the throughput by several orders of magnitude over conventional slab or capillary array systems1. Several generations of CAE microplates have been developed to optimize layout and performance. Our current design uses a circular scanning confocal fluorescence detection system together with radially symmetric channel layouts. The design consists of a common anode reservoir in the center of a circular 4" or 6" diameter wafer and an array of 96 channels extending radially outward towards injector units at the perimeter of the wafer. This radial design gives quality high speed separations by eliminating resolution reducing turns and allows the analysis of 96 samples in parallel on a single microplate. The confocal rotary scanner can measure fluorescence from DNA fragments in all channels at a rate of up to 15 samples/sec. The major advantage of a rotary scanner over linear scanners is that the motion of the scanner is continuous, making it easier to control the velocity at high sample rates. High sample rates are necessary to ensure good resolution of electrophoretic bands. The scanner is capable of collecting 2880 data points/revolution at 23.15 ms intervals. The scanner utilizes four independent ADCs to simultaneously acquire data from four color electrophoresis runs.
The operation and capabilities of the radial CAE microplates with the rotary scanning system were first demonstrated by performing high speed electrophoretic separations of 96 pBR322 MspI DNA samples in 40 seconds. Genotyping of methylenetetrahydrofolate reductase (MTHFR), a candidate gene for vascular disease and neutral tube defects, was also performed on 4'' diameter radial CAE microplates to demonstrate the rapid analysis of biologically relevant samples (in collaboration with Prof. M. Smith and C. Skibola in the School of Public Health, UCB). Two-color multiplexed fluorescence detection of the MTHFR genotypes was accomplished by prelabeling standard pBR322 MspI DNA ladder with a red emitting bisintercalation dye (butyl TOTIN) and prelabeling of the MTHFR DNA with a green emitting bisintercalation dye (TOTO)2. Using this two-color multiplexing method, 96 MTHFR DNA samples were genotyped in less than 2 minutes with 4 bp resolution. Radial CAE microplates fabricated on 6" wafers are currently being developed for ultra-high throughput DNA sequencing applications.
1P.C. Simpson, D. Roach, A.T. Woolley, T. Thorsen, R. Johnston, G. F. Sensabaugh, and R.A. Mathies, Proc. Nat. Acad. Sci., USA, 95, 2256-2261 (1998)
2S.M. Clark, and R.A. Mathies, Anal. Chem., 69, 13354-1363 (1997)
39. Integrated Sequencing Sample Preparation on CE Microplates
Yining Shi1, Indu Kheterpal1,
Jin Xie2, Alexander N. Glazer2, and Richard A. Mathies1
Microfabricated devices are revolutionizing the field of DNA electrophoresis because DNA fragments can now be separated in less than 1 minute1,2 and sequencing separations are achieved in ~10 minutes3. Furthermore, Microfabricated devices allow the integration of sample preparation, clean-up, separation and detection. To achieve this goal we have performed high quality sequencing separations on microchannels and are developing solid-phase microfluidic methods to concentrate and clean up DNA samples for efficient injection into the separation columns.
The quality of separation of DNA fragments is highly dependent on the injection and separation conditions. We have optimized four-color sequencing on microfabricated capillary electrophoretic devices for separation matrix, temperature, channel dimensions, injector size and injection parameters. Linear polyacrylamide (LPA; 4%) matrices were used to achieve sequencing separations of 600 bases on 7 cm long channels in ~20 minutes. The sequence data were analyzed and base-called using BaseFinder4 and an accuracy rate of 99.4% was obtained to 500 bases3.
We are now developing methods to integrate these excellent separations with sample preparation methods on a single device. We have synthesized biotinylated energy transfer primers for fragment amplification and sequencing5. The presence of the biotin allows us to utilize solid-phase surface chemistry to purify and concentrate DNA samples before introducing them into the separation columns. We have successfully constructed sandwich structures of biotin-streptavidin-biotin on the glass surface. The biotinylated PCR products are pumped through a capture chamber and concentrated onto the surface containing biotin-streptavidin. The products captured in the reaction chamber are cleaned, denatured with formamide at 90C and injected directly into the separation columns. These sample clean-up methods are relevant to developing fully integrated microdevices.
1 Woolley, A. T. and Mathies, R. A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11348-11352.
2 Simpson, P. C.; Roach, D.; Woolley, A. T.; Thorsen, T.; Johnston, R.; Sensabaugh, G. F. and Mathies, R. A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2256-2261.
3 Liu, S.; Shi, Y.; Ja, W.; Mathies, R. A. (1998) Anal. Chem. in press.
4 Giddings, M. C.; Severin, J; Westphall, M; Wu, J. Z; and Smith, L. M. (1998) Genome Research 8, 644-665.
5 See abstract "Synthesis, Characterization, and Potential Applications of Biotinylated Energy Transfer Oligonucleotides" by J. Xie, R. A. Mathies, A. N. Glazer.
40. Integrated Electrochemical Detection with Microfabricated Capillary Electrophoresis Chips
Pankaj Singhal1, Jin
Xie2, Alexander N. Glazer1, and Richard A. Mathies2
Microfabrication technology has enabled the development of miniaturized capillary electrophoresis (CE) chips or microdevices that can perform preparation, amplification and electrophoretic separation of a wide variety of analytes on a very short time scale1-3. However, nearly all microchip analyses to date have utilized laser excited fluorescence detection. While fluorescence detection is very effective, it is not easy to integrate the laser and optical system into the microfabricated chip to make a completely miniaturized analysis system. We have therefore been exploring the development of microfabricated electrochemical detection systems for microchip CE analyses because of the high sensitivity of this method and the ease of integration.
In our first studies, platinum electrodes were microfabricated on glass CE-chips to demonstrate the feasibility of integrated electrochemical detection4. Although, redox-active neurotransmitters were detected directly with high sensitivity, non-electroactive DNA could only be detected using indirect detection. In order to make electrochemical detection more universal for chip-based analyses, redox-active labels can be attached to inherently non-electroactive compounds. Specifically, we have synthesized an M-13 primer with hydroquinone and ferrocene labels to demonstrate the feasibility of attaching labels to DNA. We have worked out the synthetic routes to prepare active N-hydroxysuccinimide esters of these labels. Activated esters of 1, 4-dihydroxy-2-naphthoic acid or a ferrocene were coupled with a 5'-aminohexyl terminated M-13(-40) universal primer sequence to make two different electroactive DNA primers. We have been able to detect these labeled DNA primers down to zeptomole levels using our micro-fabricated CE-chips with integrated electrochemical detection.
As a number of different labels are available for attachment to various analytes, simultaneous detection of multiple samples is conceivable with very high selectivity. To demonstrate this concept, we selected various labels which exhibit different redox-properties and are therefore readily distinguishable. These labels were detected with high selectivity using CE-chips with integrated electrochemical detection. A matrix-coding method was developed to collect the electrochemical signals from each label. This method also uniquely addresses each signal, so that the labels were detected without any overlap from each other. CE-chip designs using this approach for multiplex analyses in a single separation will also be presented. To further highlight the potential of integrated electrochemical detection, we will present a fully portable version of our microchip based system. This instrument validates that integrated electrochemical detection allows CE-chip based analyses to be miniaturized and portable.
1 Woolley, A. T. and Mathies, R. A.; (1994) Proc. Natl. Acad. Sci. U.S.A. 91 11348-11352.
2 Woolley, A. T., Sensabaugh, G. F. and Mathies, R. A.; (1997) Anal. Chem. 69 2256-2261.
3 Simpson, P. C., Roach, D., Woolley, A. T., Thorsen, T., Johnston, R., Sensabaugh, G. F. and Mathies, R. A.; (1998) Proc. Natl. Acad. Sci. U.S.A. 95 2256-2261.
4 Woolley, A. T., Lao, K., Glazer, A. N. and Mathies, R. A.; (1998) Anal. Chem. 70 684-688.
41. Integrated Microchip Devices for DNA Analysis
R. S. Foote, W. C. Dunn, J. Khandurina,
N. Kroutchinina, T. McKnight, L. C. Waters, S. C. Jacobson, and J. M. Ramsey
Microfabricated microfluidic devices are being developed for integrated processing and analysis of DNA samples. The steps of DNA extraction, amplification, preconcentration and electrophoretic analysis can be carried out on monolithic devices. We have previously demonstrated integrated cell lysis, multiplex PCR and capillary electrophoretic (CE) size analysis on microchips using bacterial samples. Integrated PCR-CE microchips are now being used for the analysis of simple sequence repeat (SSR) loci in mammalian genomes. To demonstrate the potential utility of this technology for rapid PCR-based gene mapping, SSR polymorphisms (SSRPs) at two mouse genome loci, D4Mit141 and D8Mit9, were identified and compared for Mus musculus (C57/Bl or C3H), Mus spretus, a C57/Bl x spretus hybrid, and progeny from an interspecies (C3H x spretus) backcross used to create genetic maps. For both loci, microchip electrophoresis patterns of SSR fragments generated from animals heterozygous for musculus and spretus alleles were clearly distinguishable from those of homozygotes and PCR product sizes were determined for respective SSRs by co-electrophoresis with marker DNA. Integrated microchip analysis of human forensic samples has also been demonstrated by DNA fingerprinting at the CSF1PO, THO1, TPOX and vWA loci.
The microchip CE analysis time for PCR products is typically less than 5 minutes, so that the throughput for integrated PCR-CE microdevices is primarily determined by the PCR thermal cycling time. Approaches to increasing the throughput of these devices include the use of multiple reaction chambers for parallel PCR, fast thermal cycling using thermoelectric heating and cooling, and on-chip concentration of products from low cycle number PCRs. The last approach is being explored by incorporating a DNA concentration region into the microchip architecture between the reaction chamber and the separation channel. A porous membrane between two parallel channels is incorporated into the channel manifold using a silicate adhesive to bond the cover plate to the substrate. The thin silicate layer serves as a semi-permeable membrane allowing ionic current to pass between the separated channels but retaining large DNA molecules. Preconcentrated sample is then injected into the separation channel and electrophoretically analyzed. DNA fragments were concentrated on-chip from PCR amplifications by up to 2 orders of magnitude by this method, allowing product analysis at a reduced number of thermal cycles.
42. Single Nucleotide Polymorphism Detection and Identification Directly from Human Genomic DNA by Invasive Cleavage of Oligonucleotide Probes
Victor Lyamichev, Andrea L. Mast, Jeff
G. Hall, James Prudent, Tamara Sander, Monika de Arruda, David Arco, Bruce
P. Neri, and Mary Ann D. Brow
Detection of DNA by invasive cleavage arises from the coordinated action of a pair of overlapping synthetic oligonucleotides hybridized to adjacent regions on a DNA target. At the point of overlap, i.e. where one or more nucleotides from each oligomer compete for same complementary site on the target, a unique secondary structure forms when the 3' end of one oligomer (invasive probe) displaces the 5' end of the other (signal probe). This displaced "flap" is in turn recognized by a structure-specific endonuclease and cleaved to release a fragment.
The specific cleavage of a downstream flap has been employed as an extremely sensitive, quantitative, and highly specific assay for the detection of target DNA both alone and in mixture of extraneous DNA. Because cleavage depends on the correct alignment of the oligonucleotides, the cleavage is sufficiently specific to enable discrimination of single nucleotide polymorphisms (SNPs) and can readily differentiate homo- from heterozygotes in single-copy genes in human genomic DNA. Moreover, we have defined reaction conditions that allow multiple copies of the downstream oligonucleotide probe to be cleaved for each target sequence without temperature cycling, thereby amplifying the cleavage signal and allowing quantitative detection of target DNA at sub-attomole amounts.
The analysis of nucleic acids in this fashion has several advantages over existing methods of oligonucleotide-based detection. First, by requiring two oligonucleotides, the reaction is highly specific for the intended target sequence. Second, the specificity of the enzyme requires precise alignment of the probes for cleavage to occur, providing a much higher level of specificity than can be achieved by hybridization alone, and allowing single-base discrimination of multiple alleles present in a mixed sample. Third, the products of this cleavage reaction can be analyzed via indirect readouts that utilize the nucleotide sequence of products, such as by capture on solid supports, thus simplifying the equipment needed to perform the procedure and adding yet another level of discrimination for the desired cleavage products. Fourth, amplification of a target-dependent signal, rather than the target itself, means that traces of product "carried over" from a completed detection reaction cannot themselves be amplified to lead to false positive results. Finally, detection of specific sequences directly from genomic DNA without intervening DNA amplification avoids false negative or false positive SNP detection that may arise due to low fidelity replication during an amplification step.
43. High Throughput SNP Discovery and Scoring Using Bead-Based Flow Cytometry
P. Scott White, Hong Cai, and John
There is a pressing need for SNP discovery and analysis capabilities that are rapid and robust. We are developing approaches using microsphere-based flow cytometry to address these needs.
For SNP discovery, we have developed a system that uses immobilized mismatch-binding proteins (IMBP) to detect SNPs in heteroduplexes. IMBP-coated microspheres are added to fluorescently labeled PCR amplicons, and analyzed by flow cytometry. The detection of fluorescence associated with the beads indicates the amplified region contains one or more SNPs, which are then sequenced.
Bead-based minisequencing or oligo ligation using flow cytometry is used to score SNPs. A novel system for multiplexed analysis enables simultaneous scoring of 64 or many more different SNPs/sample. Furthermore, because of the quantitative nature of flow cytometry, pooling amplicons from large numbers of individuals will allow for the determination of the frequencies of each SNP in populations.
These microsphere-based flow cytometric analyses have the following general advantages: 1) Intrinsic resolution between free and microsphere-bound probe, allowing homogeneous assays with no wash steps; 2) Quantitative, multicolor fluorescence detection with sensitivity surpassing microplate or microscope-based detection; 3) Soluble solid phase that can be prepared, pipetted, and handled by conventional fluidics systems; and 4) An instrument that is already available in core facilities at the vast majority of research universities, medical schools, pharmaceutical companies, and clinical diagnostic laboratories. Furthermore, the potential for multiplexing these assays will greatly enhance throughput and allow for the scanning of over one megabase/day for new SNPs, or for scoring thousands of individuals for hundreds to thousands of known SNPs/day.
44. DNA Characterization by Electrospray Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
David S. Wunschel, Ljiljana Pasa Tolic,
Bingbing Feng, James E. Bruce, Harold R. Udseth, and Richard D. Smith
Mass spectrometry offers the potential for high speed DNA sequencing and ultra-sensitive characterization. Ongoing work in the laboratory is exploring approaches based upon electrospray ionization (ESI) and/or Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. These efforts have included advanced methods for the characterization of polymerase chain reaction (PCR) products 1, enzymatically produced oligonucleotide mixtures, modified DNA and the development of methods for the analysis of DNA large fragments. High mass accuracy measurements for PCR products allowing a single base substitutions to be detected at the 250 bp level with de novo identification of an unreported base substitution. This capability also allows the identification of small differences in mass such as those arising from methylation2. Study of DNA damage/modifications in their sequence context will likely have to occur from within multi-component mixtures. The capability for this has been demonstrated using a multi-component reaction where a base pair deletion was identified with the putative identification of inter-operon variability within a single bacterial strain3. These efforts are also being extended to exploit the non-destructive nature of FTICR for recovery (i.e., "soft-landing") of mass-selected modified DNA segments, following high resolution FTICR analysis and separation (i.e., high resolution sorting), for subsequent cloning or PCR. This would allow for direct selection and analysis of individual components from within mixtures that may share a high degree of similarity without cloning. Alternatively, DNA species that cannot be identified through traditional sequencing methodologies, those containing base modifications, can be isolated with the nature and position of the modification identified. Most importantly this potentially allow identification of low abundance products containing modifications where few if any alternatives for their detection exist. These and related recent advances will be described.
1 "Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry", D. C. Muddiman, D. S. Wunschel, C. L. Liu, L. Pasa Tolic, K. F. Fox, A. Fox, G. A. Anderson and R. D. Smith, Anal. Chem. 68, 3705-3712 (1996)
2 "Mass measurement of a PCR product from the Lambda bacteria phage at the 223 base pair level by ESI-FTICR", D. S. Wunschel, B. Feng, L. Pasa Tolic, and R. D. Smith.
3 "Heterogeneity in Bacillus cereus PCR Products Detected by ESI-FTICR Mass Spectrometry", D. S. Wunschel, D. C. Muddiman, K. F. Fox, A. Fox, and R. D. Smith, Anal. Chem. 70, 1203-1207 (1998)
This research was supported by the Office of Biological and Environmental Research, U.S. Department of Energy. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute through Contract No. DE-AC06-76RLO 1830.
45. Laser Desorption Mass Spectrometry for DNA Sequencing and Analysis
C. H. Winston Chen, N. R. Isola,
N. I. Taranenko, V. V. Golovlev, and S. L. Allman
During the past few years, rapid progress has been achieved for both slab gel electrophoresis and capillary gel electrophoresis. Many experts in the field expect that most parts of human genome can be sequenced within next 3 to 7 years. However, some portions of DNA in human genome which has long repeats and with secondary hairpin structures are still very difficult to be sequenced by conventional gel electrophoresis with Sanger's enzymatic method to produce DNA ladders. The band compression often occur for DNA segments with high GC ratio and/or with secondary structures. PCR process may not faithfully replicate the DNAs which have hair pin structures. Thus, a new and reliable approach to sequence these "difficult" templates is critical for completing the sequencing of the entire human genome. Recently, we tried to couple laser desorption mass spectrometry with Maxam Gilbert chemical degradation method to produce DNA ladders to achieve sequencing of DNA templates with high GC component. DNA templates were first bound with biotin so that DNA ladders produced by the chemical degradation method can be isolated from the solution by magnetic bead streptavidin separation. Then these isolated DNA segments are released from streptavidin and subsequently analyzed by laser desorption mass spectrometry. Since the sequencing by laser desorption mass spectrometry is based on the measurement of molecular weights, band compression is no longer a problem. Since no PCR is required, non-faithful replication by PCR due to the secondary structures or a large number of repeat can be eliminated.
In addition to the sequencing by Maxam Gilbert's approach, we also used laser desorption mass spectrometry for DNA sequencing for DNA ladders produced by Sanger's method. ss-DNA templates larger than 100 nt were successfully sequenced. ds-DNAs larger than 200 bp were also sequenced. However, mass resolution and detection sensitivity still need more improvement for sequencing longer DNAs. In addition to sequencing DNA with ladders produced by chemical methods, we also developed a technology to produce DNA ladders during the laser desorption process. By controlling the pH value and selecting the right matrices, direct sequencing of short DNA was obtained without the need of the preparation of DNA ladders. Since the sequencing process with this approach is very fast, it can be used to sequence a large number of probes which are often used for diagnosis by hybridization.
* Research has been supported by DOE/OBER
46. PCR Product Size Measurement using MALDI Mass Spectrometry
G.B. Hurst, Y. Kim, K. Weaver, and
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has considerable potential as a technique for rapid and accurate analysis of PCR products. We are pursuing two specific end-uses of this technique: mapping mutant phenotypes to chromosome regions and determining the extent of chromosomal rearrangements that are targets for mutagenesis in collaboration with ORNL's Laboratory for Comparative and Functional Genomics and screening of endogenous bacterial to assess genetic potential for bioremediation, in collaboration with Mary Lidstrom at the University of Washington. Current capabilities include virtually routine analysis with near single-base resolution up to 100 bases, and the less routine ability to measure 200-mers or larger products. We are working to further extend this technique to larger PCR products (and similarly-sized DNA derived from other sources), as well as to develop schemes for scaling up the applicability of MALDI to larger numbers of samples.
The salts and buffers necessary as reagents for the PCR act as interferences for the MALDI process, and therefore must be removed prior to MALDI-MS analysis. To overcome this problem, we have developed a rapid reverse-phase method for purifying PCR products 1, and have demonstrated the parallel implementation of this procedure in a 96-well microtiter-format using a filter plate loaded with the appropriate reverse-phase resin. Manual implementation of the 96-well purification method, using an 8-channel pipettor and a vacuum manifold, requires approximately 20-30 minutes for 96 samples.
For MALDI-MS analysis, PCR products must be combined with a matrix material and allowed to dry on a sample plate. The resulting inhomogeneous spot is sparsely dotted with regions that yield useful spectra when interrogated with the desorption laser ("sweet spots"), and therefore requires either human expertise for laboriously choosing promising locations across the sample, or inefficient automated positioning of the laser at numerous positions in hopes of locating a sweet spot. For this reason, we are developing methods for preparing more homogeneous spots containing the mixture of PCR product and matrix, using polymeric substrates and/or additives. Fluorescently-labeled primers or PCR products may allow us to correlate MALDI-MS results with fluorescence microscopy imaging of the DNA distribution in these samples.
1 "MALDI-TOF Analysis of Polymerase Chain Reaction Products from Methanotrophic Bacteria," G.B. Hurst, K. Weaver, M.J. Doktycz, M.V. Buchanan, A.M. Costello, and M.E. Lidstrom, Anal. Chem. 70, 2693-2698 (1998).
Research supported by the Environmental Management Science Program, Office of Biological and Environmental Research, U.S. Department of Energy, and the Oak Ridge National Laboratory Director's Research and Development Funds. Oak Ridge National Laboratory is managed for the United States Department of Energy by Lockheed Martin Energy Research Corp. under contract DE-AC05-96OR22464.
47. Analyzing Genetic Variations by Mass Spectrometry
Lloyd M. Smith, Travis Berggren,
Tim Griffin, Zhengdong Fei, and Mark Scalf
In the last decade two powerful new tools for the mass spectrometric analysis of biomolecules have been developed, Matrix-Assisted Laser Desorption Mass Spectrometry (MALDI-MS), and Electrospray Ionization Mass Spectrometry (ESI-MS). The power of these methods lies in their ability to produce and mass analyze intact gas phase ions from very large molecules such as proteins and nucleic acids. The speed, accuracy, and sensitivity of the technologies make them well-suited to address a number of problems in genetic analysis, including the analysis of DNA sequence, genetic variations, and gene expression. Results in these areas will be presented, including recent work in which single nucleotide polymorphisms (SNPs) in genomic DNA may be analyzed without need for a prior PCR amplification step.
48. DNA Sequencing by Single Molecule Detection
James H. Jett, Peter M. Goodwin,
James H. Werner, Hong Cai, and Richard A. Keller
We are developing a DNA sequencing technique that is based upon single fluorescent molecule detection. The goal is to sequence long strands of DNA approaching 40 kb in length at rates of 50 bases per second. The approach being pursued is to: 1) fluorescently label a strand of DNA by enzymatic incorporation of prelabeled nucleotides; 2) attach a single labeled strand of DNA to a microsphere; 3) suspend the microsphere in the flow stream of a flow cytometer capable of single molecule detection and identification; 4) add an exonuclease with activating cofactors that will cleave sequentially the labeled nucleotides; and 5) identification of the cleaved nucleotides by analysis of laser-induced fluorescence from the label attached to the nucleotides. We have made considerable progress towards demonstrating this approach to DNA sequencing. Briefly, the current status of each of the steps above is as follows. Up to three base types in strands of DNA 2-7 kbp long have been labeled base identifying fluorophors. Multiple strands of labeled DNA have been attached to microspheres and individual microspheres suspended by a laser optical trap in the flow stream of the detection apparatus. Enzymatic activity of several exonucleases on DNA attached to microspheres under flowing conditions has been observed. Fluorescent molecule identification at the single molecule level has been demonstrated by correlated measurements of fluorescent burst intensity and fluorescence lifetime with a single excitation wavelength and a single detection channel. Details of the progress made in each of the steps will be discussed.
This work is supported by the US Department of Energy, Office of Biological and Environmental Research.
49. Manipulation of Single DNA Molecules by Induced-Dipole Forces in Micro-Fabricated Structures
Chip Asbury, Paolo Prati, and Ger
van den Engh
We are exploring the use of induced-dipole forces in oscillating, divergent electric fields, for trapping, moving and stretching DNA molecules. These forces, which are distinct from electrophoretic forces, can be generated by means of microscopic metal patterns on quartz substrates. Micro-fabrication techniques can be employed to generate large numbers of traps on a single wafer. This technology lends itself well for massive automation and parallelization of DNA sample preparation.
Unlike electrodes for electrophoresis, DNA trapping can be achieved with floating electrodes. Voltage applied by external wires to the fluid is passively distributed among hundreds of microelectrodes. The electric field lines concentrate on the electrode edges exerting strong attractive forces on DNA molecules in solution. If the electrode gaps are small, significant trapping forces can be obtained without inducing electrolysis. The magnitude of these forces appears to vary with DNA molecular weight.
We have built several devices for manipulating small quantities of DNA, such as shift registers, DNA concentrators, etc.. We are now developing more complex structures that combine DNA dipole traps with more elaborate manipulations. We have made electrophoresis capillaries with concentrating traps at the entrance and exit. We have constructed capillaries with a series of traps along their length for size dependent DNA separation. We are working on a device that guides DNA molecules along a precise trajectory past a fluorescence detector. We are also exploring the use of traveling waves to concentrate DNA from a large area.
We will describe these and other structures and will present the conditions under which these devices are most efficiently used.
50. A Quantitative Analytical Tool for Improving DNA-Based Diagnostic Arrays
Tom J. Whitaker
Sequence analysis using hybridization on ODN arrays is particularly well suited for genetic diagnostics, sequencing cDNAs, and partial sequencing of clones to allow mapping. In spite of this, quality control issues have hampered the full acceptance of these arrays, which are often called "gene chips". A high dynamic range, quantitative measurement method is needed to study parameters that increase efficiency and new methods of array production. With such a tool, systematic studies of hybridization strategies could be undertaken which would almost certainly lead to improved efficiencies and lower array manufacturing costs. Although fluorescence detection has been adequate for analysis of the hybridized chips, it does not have the spatial resolution or dynamic range to image the surface density of small (e.g. 20m spot size) bound probe ODNs or to perform hybridization kinetics studies.
We have just begun a new project to develop and utilize a high-resolution, quantitative method to analyze ODNs on an array. The technique involves detection of tin-labeled ODNs by sputtering them from the surface with an energetic ion beam, selectively ionizing the resulting tin atoms with wavelength-tunable lasers, and analyzing the ions with time-of-flight mass spectrometry. We have previously shown that this sputter-initiated resonance ionization microprobe (SIRIMP) technique can have sub-mm resolution and is highly quantitative in measuring a wide range of concentrations of elements in semiconductors. We have also shown that SIRIMP can detect DNA fragments labeled with stable isotopes 1,2. We now plan to apply SIRIMP to measurements of tin-labeled ODNs immobilized on a surface and to tin-labeled ODNs synthesized in situ on the surface. The initial phases will be used to develop and demonstrate the technique and calibration procedures. In later stages, we will work closely with Affymetrix to analyze and image in situ arrays with very small (20m) features to determine the homogeneity of binding. Additionally, hybridization experiments will be performed with two different stable isotopes of tin labeling the probe and target ODNs to determine the correlation between the surface density of the immobilized probes and hybridized targets.
The research reported here was funded, in whole or in part, by DOE grant #DE-FG02-98ER82536. Such support does not constitute an endorsement by DOE of the views expressed in this abstract.
1H.F. Arlinghaus, M.N. Kwoka, X.-Q. Guo and K.B. Jacobson, Analytical Chemistry 69, 1510 (1997).
2H.F. Arlinghaus, M.N. Kwoka, and K.B. Jacobson, Analytical Chemistry 69, 3747 (1997).
51. A Light-Directed DNA/RNA- Microarray Synthesizer
Xiaochuan Zhou1, Robert
Setterquist1, Xiaolian Gao2, Peilin Yu2,
Eric LeProust2, Laëtitia Sonigo2, Jean Philippe
Pellois2, Hua Zhang2, Erdogan Gulari3,
and Ning Gulari3
Practical advancement in biochip technologies for routine use in drug discovery and genomic applications will require a flexible and affordable chip-fabrication technology. To address this need, a multidiscipline project has been initiated. A programmable, light-directed DNA/RNA array synthesizer that uses solution-based photochemical synthesis is being developed for efficient production of high-density DNA/RNA chips. This presentation reports on the latest results of instrument development and photochemistry effort.
The project consists of three main integrated tasks: (1) design and construction of a programmable photolithographic system, (2) development of a novel solution photochemistry for nucleic acid synthesis, and (3) design and fabrication of synthesis microreactors. At the heart of the photolithographic system is a commercially available digital spatial optical modulator, which accurately produces light patterns that are used for initiating parallel high-density nucleic acid synthesis. The digital spatial optical modulator effectively replaces the necessity for using photomasks technologies. The solution photochemistry under investigation is a modification of well-established conventional synthesis protocols. Microreactors are being developed using standard microfabrication processes in order to implement the DNA/RNA synthesis photochemistry. Each reactor contains an array of microfabricated reaction wells (synthesis sites). Each microwell serves to individually isolate each reaction during the light-directed parallel sequence syntheses.
The outcome of the undertaken project will lead to a prototype DNA/RNA array synthesizer. The prototype instrument will be further developed into a commercial model. The envisioned instrument will allow researchers to make high-density and high fidelity DNA/RNA-chips of their own designs at an affordable cost. In addition, there is obvious potential to expand the light-directed chemical approach in this project for synthesis of other combinatorial arrays (peptide, carbohydrate, and small molecule).
52. Development of Flowthrough Genosensor Chips
Mitchel J. Doktycz and Kenneth L.
A flowthrough genosensor chip is under development at ORNL. The core of this technology is a microchannel hybridization array, containing numerous specific DNA sequences, immobilized within individual cells of densely packed straight, smooth channels traversing a thin silicon or glass substrate. When a nucleic acid sample is labeled and passed through the microchannel genosensor chip, hybridization occurs at porous cells bearing immobilized DNA probes complementary to the target sequence. The quantitative binding pattern reflects the relative abundance of specific target sequences within the nucleic acid analyte. The flowthrough chip configuration has several important advantages over flat surface DNA chips being developed elsewhere: faster hybridization kinetics, superior binding capacity, improved ability to analyze dilute solutions of nucleic acids, including both strands of a heat-denatured PCR fragment.
Related technology for taking advantage of the benefits of the flowthrough genosensor include the development of micromachining techniques for the construction of flowthrough silicon chips to complement those constructed using channel glass. A customized robotic spotting system has been developed that includes a high resolution positioning system, sapphire dispensing tips for touch-off dispensing, and, more recently, solenoid-controlled ink jets for remote droplet delivery. A prototype fluidics system has been developed that involves syringe pump-driven fluid flow, a custom chip holder attached to the stage of a Zeiss Axiovert fluorescence microscope and a CCD camera for real-time quantitative detection of hybridized fluorescent-labeled strands. A software package for intelligent selection of oligonucleotide probes for a given chip application has been developed.
The flowthrough genosensor system is now being used to develop applications in the areas of genotyping and mRNA profiling, in collaboration with various laboratories. Gene expression profiling in mammalian systems, including mouse and sheep, is being pursued as well as bacterial systems for evaluating expression patterns in soil microorganisms as an indicator of genotoxic response in the environment. Another application being developed is high throughput genotyping. In this work miniature flowthrough genosensors are used to simultaneously analyze numerous single nucleotide and short insertion-deletion polymorphisms. In another application area, the ultrahigh surface area of channel glass is being exploited to create arrays of "microreactor cells" containing immobilized BAC DNAs, for use in repetitive reactions needed for genome mapping and sequencing, including cycle sequencing reactions, PCR, and hybridization mapping of expressed sequences to their genomic clones.
53. Sequence Analysis and Thermodynamic Studies of Short DNA Duplexes on Oligonucleotide Generic Microchip
A. Fotin, D. Proudnikov, E. Timofeev,
G. Yershov, Eu. Kirillov, A. Drobyshev, E. Khomyakova, A. Zasedatelev,
and A. Mirzabekov
Generic microchip -- an oligonucleotide microchip containing a complete set of hexanucleotide probes -- has been manufactured and applied for sequence analysis and thermodynamic studies. Isothermal hybridizations as well as thermal denaturation experiments allowed performing sequencing of model synthetic oligomers up to 70 bases long. Melting experiments on the chip using fluorescent microscope have demonstrated reliable discrimination between perfect and mismatched duplexes. This technique was succesively applied for identification of mutations. Studies of thermodynamics of DNA duplex and triplex formation and the effect of modified bases and minor groove binding ligands on duplex stability have been carried out on the generic microchip.
53a. Mass Spectrometry for Analyzing and Sorting DNA Ions
W. Henry Benner and Joseph M. Jaklevic
The development of new ion detectors for mass spectrometry is the focus of this project. Our recent developments of charge- and energy- sensitive ion detectors offer new ways to measure and manipulate individual DNA molecules. Charge detection mass spectrometry (CDMS), invented at LBNL, measures the molecular weight of DNA molecules larger than about 500 bp. Starting with electrospray ionization for generating highly charged DNA ions, CDMS measures the charge and velocity of individual ions in vacuum as they fly through a small metal tube. The mass distribution of the molecules in a sample is calculated from measurements made on a number of ions. Typically, only about 1000 ions are needed to produce a peak in a mass spectrum for each component in the sample. We have demonstrated that CDMS can be used to analyze mixtures of linear ssDNA and plasmid DNA in the 1 to 15 kbp range. The upper mass limit extends well beyond this range. WE will show how CDMS is well suited for rapidly measuring the size of DNA fragments generated in the course of sequence verification. Our estimates predict that optimized signal processing will provide a way to analyze about 200 ions per second with CDMS. At this rate, sequence verification performed on RE digests of BACs could be performed more rapidly than with pulsed-field gel separations. Since CDMS performs a nondestructive measurement on DNA ions, it opens the possibility for sorting individual molecular ions. A device has been designed for sorting individual ions into the wells of a microtiter plate for the purpose of performing single molecule studies. Our sorting device works similarly to a flow sorter but operates on the principe of selection based on ion mass. We have also developed an energy-sensitive ion detector called a superconducting-tunnel junction (STJ). Our STJ detector responds to the energy deposited by an impacting ion. This ion detector has helped to extend the efficiency for detecting high mass ions in applications related to time-of-flight mass spectrometry. Now that IR-MALDI appears capable of ionizing large DNA molecules, this detector will be used to detect DNA ions in sequencing ladders by means of IR-MALDI-MS.
(This work was supported by the Director, Office of Energy Research, Office of Health and Environmental Research, Human Genome Program, U.S. Department of Energy under contract number DE-AC03-76SF00098.)