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| Sequencing
Part I Index |
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An Engineered RNA/DNA Polymerase to Increase Speed and Economy of
DNA Sequencing
DNA sequence information is the cornerstone for considerable experimental design and analysis in the biological sciences. The proposed studies will focus on advancing DNA sequencing by creating a new enzyme that eliminates the need for an oligonucleotide primer to initiate DNA synthesis at a defined site, and that can use dideoxy nucleotides for chain termination. The new method should reduce the time and cost required to obtain DNA sequences and enhance the speed and cost effectiveness of current DNA sequencing technologies. Phase I studies will focus on purifying mutant T7 RNA polymerases known to incorporate dNTPs into DNA chains, developing protocols for rapid small scale mutant enzyme purification, evaluating the purified mutants for properties relevant to DNA sequencing, developing facile mutagenesis schemes and producing mutant RNA/DNA polymerases with altered promoter recognition. The results from phase I will provide the foundation for Phase II research, which will focus on refining properties of the mutant by: (1) expanding the number of mutations examined using the purification protocols, assays, and mutagenesis screening methods developed in Phase I and (2) examining the effect of each mutation on enzymatic properties important to DNA sequencing applications, and (3) optimizing conditions for sequencing performance. In Phase III, Promega will commercialize the new mutant enzymes through its own extensive distribution network and by collaborating with major instrumentation firms to adapt the technology to automated DNA sequencing systems. DOE Grant No. DE-FG02-96ER8226.
Directed Multiple DNA Sequencing and Expression Analysis by Hybridization Gualberto Ruano BIOS Laboratories, Inc.; New Haven, CT 06511 800/678-9487 or 203/773-1450, Fax: 800/315-7435 or 203/562-9377 The overall goal of this project is to develop molecular resources with direct applications to either DNA sequence analysis or gene expression analysis in multiplexed formats using sequential hybridization of Peptide Nucleic Acid (PNA) oligomer probes. PNA oligomers hybridize more stably and specifically to cognate DNA targets than conventional DNA oligonucleotides. The Phase I project discussed here is concerned with development of PNA probe technology having direct application either to the directed sequencing process or to gene expression profiling. With regard to directed sequencing, we seek improvements in the three multiply repeated steps associated with this process, namely (1) probe assembly, (2) sequencing reactions, and (3) gel electrophoresis. In PNA hybridization sequencing, sequences are generated directly from the template by multiplex DNA sequencing using anchor primers known to have frequent annealing sites. Electrophoresis is performed en masse for each anchor primer reaction, blotted to nylon membranes and individual sequences are selectively exposed by iterative hybridization to specific 8-mer PNA probes derived from sequences statistically over-represented in expressed DNA and obtained from a pre-synthesized library. Additionally, the same PNA library can be used as a source of hybridization probes for querying expression patterns of specific genes in any cell line or tissue. Specific gene expression can be monitored by coupling gene-specific RT-PCR with hybridization when cDNA products are separated by gel electrophoresis and blotted to nylon membranes. Patterns of gene expression are then resolved by hybridization using PNA oligomers. Bands corresponding to specific genes can be deconvoluted using sequence information from RT-PCR primers and PNA probes. Higher throughput expression analysis can be achieved by multiplexed gel electrophoresis, blotting and iterative probing of RT-PCR reactions with individual PNA probes. DOE Grant No. DE-FG02-96ER8213.
1996 Phase II A Graphical Ad Hoc Query Interface Capable of Accessing Heterogeneous
Public Genome Databases
The interoperability of public genome databases is expected to be crucial in making the Human Genome Project a success. This project will develop software tools in which users in the genome community can learn or examine public genome database schemes in a relatively short time and can produce a correct Structured Query Language (SQL) expression easily. In Phase I, a concept system was constructed and the effectiveness of formulating ad hoc queries graphically was demonstrated. Phase II will focus on transforming the concept system into a product that is robust and portable. Two types of computer programs will be developed. One is a client program which is to be distributed to community users who intend to access public genomic databases and link them with local databases. The other is a server program and a suite of software tools designed to be used by those genome centers which intend to make their databases publicly accessible. DOE Grant No. DE-FG02-95ER81906.
Low-Cost Automated Preparation of Plasmid, Cosmid, and Yeast DNA Tuyen Nguyen, RandyF. Sivila, JoshuaP. Dyer, and William P. MacConnell MacConnell Research Corporation; San Diego, CA 92121 619/452-2603, Fax: -6753 MacConnell Research currently manufactures and sells a low cost automated bench-top instrument that can purify up to 24 samples of plasmid DNA simultaneously in one hour at a cost of $0.65 per sample and under $8000 for the instrument. The patented instrument uses a form of agarose gel electrophoresis to purify the plasmid DNA and electroelutes into approximately a 20 +l volume. The instrument has many advantages over other robotic and manual methods including the fact that is it two times faster, at least six times less expensive, much smaller in size, easier to operate, less cost per sample, and results in DNA pure enough for direct use in fluorescent automated sequencing. The instrument process begins with bacterial culture which is loaded directly into a disposable cassette in the machine. In Phase II work we are developing an instrument which simultaneously
purifies plasmid DNA from up to 192 (2 X 96) bacterial samples in 1.5 hours.
Prototypes of this instrument thus far constructed have allowed the purification
of 37 micrograms of high purity plasmid DNA per lane from 1.5 ml of
bacterial culture. We have attempted to optimize all of the: instrument
electrophoretic run parameters, lysis chemistry, lysis reagent delivery
devices, reagent storage at room temperature, desalting processes and overall
instrument mechanical and electronic control. Instrument prototypes have
also been used to prepare cosmid or yeast DNA in quantities of 15
micrograms per cassette lane. Trials thus far have yielded plasmid DNA
of sufficient purity for direct use in automated fluorescent and manual
sequencing as well as other molecular biology protocols. We have studied
the purity of the resulting DNA when directly sequenced on a Licor 4000
Long Reader and ABI 373A automated DNA sequencers. Results from the Licor
4000 instrument give routine read lengths of >850 base pairs with 98% accuracy
while ABI 373A reads generally exceed 400 base pairs with similar accuracy.
DOE Grant No. DE-FG03-94ER81802/A000.
GRAIL-GenQuest: A Comprehensive Computational Framework for DNA Sequence Analysis Ruth Ann Manning ApoCom, Inc.; Oak Ridge, TN 37830 423/482-2500, Fax: /220-2030 Although DNA sequencing in the Human Genome Project is occurring fairly systematically, biotechnology companies have focused on sequencing regions thought to contain particular disease genes. The client-server DNA sequence analysis system GRAIL is the most accurate and widely used computer-based system for locating and characterizing genes in DNA sequences, but it is not accessible to many biotechnology environments. The GRAIL client software and graphical displays have been developed for high-end UNIX-based computer workstations. Such workstations are standard equipment in universities and large companies, but personal computers (PCs) and Macintosh computers are the prevalent technology within the biotechnology community. This PhaseI project will design Macintosh- and Windows-based client graphical user interface prototypes for GRAIL. The growth of DNA databases is expected to continue at a fast pace in
the attempt to sequence the human genome completely by the year 2005. Parallel
processing is a viable solution to handle searching through the ever-increasing
volume of data. During PhaseI, genQuestthe sequence comparison server portion
of the GRAIL systemwill be parallelized for shared-memory platforms and
will use PVM1 for the development of genQuest servers on networks
of PCs and workstations and other innovative, high-performance computer
architectures.
1The Parallel Virtual Machine (PVM) message-passing library
allows a collection of UNIX-based computers to function as a single multiple-processor
supercomputer.
Note: The proceedings of the 1997 DOE
Human Genome Program Contractor-Grantee Workshop VI, which include updated
research abstracts, can be found at:
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