Lawerence Berkeley National Laboratory
Human Genome Center
In lieu of individual
research projects and investigators
at the LANL Center for Human Genome
Studies are represented in
this narrative. More information can be found on the center's Web
1937 the Ernest Orlando
Lawrence Berkeley National Laboratory (LBNL) has been a major contributor
to knowledge about human health effects resulting from energy production
and use. That was the year John Lawrence went to Berkeley to use his brother
Ernest's cyclotrons to launch the application of radioactive isotopes in
biological and medical research. Fifty years later, Berkeley Lab's Human
Genome Center was established.
Now, after another decade, an expansion of biological research relevant to Human Genome Project goals is being carried out within the Life Sciences Division, with support from the Information and Computing Sciences and Engineering divisions. Individuals in these research projects are making important new contributions to the key fields of molecular, cellular, and structural biology; physical chemistry; data management; and scientific instrumentation. Additionally, industry involvement in this growing venture is stimulated by Berkeley Lab's location in the San Francisco Bay area, home to the largest congregation of biotechnology research facilities in the world.
In July 1997 the Berkeley genome center became part of the Joint Genome Institute.
Large-scale genomic sequencing has been a central, ongoing activity at Berkeley Lab since 1991. It has been funded jointly by DOE (for human genome production sequencing and technology development) and the NIH National Human Genome Research Institute [for sequencing the Drosophila melanogaster model system, which is carried out in partnership with the University of California, Berkeley (UCB)]. The human genome sequencing area at Berkeley Lab consists of five groups: Bioinstrumentation, Automation, Informatics, Biology, and Development. Complementing these activities is a group in Life Sciences Division devoted to functional genomics, including the transgenics program.
The directed DNA sequencing strategy (see below, left) at Berkeley Lab was designed and implemented to increase the efficiency of genomic sequencing. A key element of the directed approach is maintaining information about the relative positions of potential sequencing templates throughout the entire sequencing process. Thus, intelligent choices can be made about which templates to sequence, and the number of selected templates can be kept to a minimum. More important, knowledge of the interrelationship of sequencing runs guides the assembly process, making it more resistant to difficulties imposed by repeated sequences. As of July 3, 1997, Berkeley Lab had generated 4.4 megabases of human sequence and, in collaboration with UCB, had tallied 7.6 megabases of Drosophila sequence.
Instrumentation and Automation
Recent advances in the instrumentation group include DNA Prep machine and Prep Track. These instruments are designed to automate completely the highly repetitive and labor-intensive DNA-preparation procedure to provide higher daily throughput and DNA of consistent quality for sequencing (see photo below and at left).
Berkeley Lab's near-term needs are for 960 samples per day of DNA extracted
from overnight bacteria growths. The DNA protocol is a modified boil prep
prepared in a 96-well format. Overnight bacteria growths are lysed, and
samples are separated from cell debris by centrifugation. The DNA is recovered
by ethanol precipitation.
The informatics group is focused on hardware and software support and system administration, software development for end sequencing, transposon mapping and sequence template selection, data-flow automation, gene finding, and sequence analysis. Data-flow automation is the main emphasis. Six key steps have been identified in this process, and software is being written and tested to automate all six. The first step involves controlling gel quality, trimming vector sequence, and storing the sequences in a database. A program module called Move-Track-Trim, which is now used in production, was written to handle these steps. The second through fourth steps in this process involve assembling, editing, and reconstructing P1 clones of 80,000 base pairs from 400-base traces. The fifth step is sequence annotation, and the sixth is data submission.
Annotation can greatly enhance the biological value of these sequences. Useful annotations include homologies to known genes, possible gene locations, and gene signals such as promoters. LBNL is developing a workbench for automatic sequence annotation and annotation viewing and editing. The goal is to run a series of sequence-analysis tools and display the results to compare the various predictions. Researchers then will be able to examine all the annotations (for example, genes predicted by various gene-finding methods) and select the ones that look best.
Nomi Harris developed Genotator, an annotation workbench consisting of a stand-alone annotation browser and several sequence-analysis functions. The back end runs several gene finders, homology searches (using BLAST), and signal searches and saves the results in ".ace" format. Genotator thus automates the tedious process of operating a dozen different sequence-analysis programs with many different input and output formats. Genotator can function via command-line arguments or with the graphical user interface.
|Progress to Date
· 40-megabase nonchimeric map containing 82 yeast artificial chromosomes (YACs) in the chromosome 5 distal long arm.
· 20-megabase contig map in the region of 5q23-q33 that contains 198 P1s, 60 P1 artificial chromosomes, and 495 bacterial artificial chromosomes (BACs) linked by 563 sequenced tagged sites (STSs) to form contigs.
· 20-megabase bins containing 370 BACs in 74 bins in the region of 5q33-q35.
· 186 clones isolated in the major DS region of chromosome 21 comprising about 3megabases of genomic DNA extending from D21S17 to ETS2. Through cross-hybridization, overlapping P1s were identified, as well as gaps between two P1 contigs, and transgenic mice were created from P1 clones in the DS region for use in phenotypic studies.
Divining landmarks for human disease amid the enormous plain of the human genetic map is the mission of an ambitious partnership among the Berkeley Lab; University of California, San Francisco; and a diagnostics company. The collaborative Resource for Molecular Cytogenetics is charting a course toward important sites of biological interest on the 23 pairs of human chromosomes.
The Resource employs the many tools of molecular cytogenetics. The most basic of these tools, and the cornerstone of the Resource's portfolio of proprietary technology, is a method generally known as "chromosome painting," which uses a technique referred to as fluorescence in situ hybridization or FISH. This technology was invented by LBNL Resource leaders Joe Gray and Dan Pinkel.
A technology to emerge recently from the Resource is known as "Quantitative DNA Fiber Mapping (QDFM)." High-resolution human genome maps in a form suitable for DNA sequencing traditionally have been constructed various methods of fingerprinting, hybridization, and identification of overlapping STSs. However, these techniques do not readily yield information about sequence orientation, the extent of overlap of these elements, or the size of gaps in the map. Ulli Weier of the Resource developed the QDFM method of physical map assembly that enables the mapping of cloned DNA directly onto linear, fully extended DNA molecules. QDFM allows unambiguous assembly of critical elements leading to high-resolution physical maps. This task now can be accomplished in less than 2days, as compared with weeks by conventional methods. QDFM also enables detection and characterization of gaps in existing physical mapsa crucial step toward completing a definitive human genome map.
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