Resources for Gene Discovery
Hunting for disease genes is not a specific goal of the DOE Human Genome
Program. However, DOE-supported libraries sent to researchers worldwide
have facilitated gene hunts by many research teams. DOE libraries have
played a role in the discovery of genes for cystic fibrosis, the most common
lethal inherited disease in Caucasians; Huntington's disease, a progressive
lethal neurological disorder; Batten's disease, the most prevalent neurodegenerative
childhood disease; two forms of dwarfism; Fanconi anemia, a rare disease
characterized by skeletal abnormalities and a predisposition to cancer;
myotonic dystrophy, the most common adult form of muscular dystrophy; a
rare inherited form of breast cancer; and polycystic kidney disease, which
affects an estimated 500,000 people in the United States at a healthcare
cost of over $1 billion per year.
The team led by Fa-Ten Kao (Eleanor Roosevelt Institute) has microdissected
several chromosomes and made derivative clone libraries broadly available
to disease-gene hunters. This resource played a critical role in isolating
the gene responsible for some 15% of colon cancers.
Of Mice and Humans: The Value of Comparative
Analyses
A remaining challenge is to recognize and discriminate all the functional
constituents of a gene, particularly regulatory components not represented
within cDNAs, and to predict what each gene may actually do in human biology.
Comparing human and mouse sequences is an exceptionally powerful way to
identify homologous genes and regulatory elements that have been substantially
conserved during evolution.
Researchers led by Leroy Hood (University of Washington, Seattle) have
analyzed more than one million bases of sequence from T-cell receptor (TCR)
chromosome regions of both human and mouse genomes. Many subtle functional
elements can be recognized only by comparing human and mouse sequences.
TCRs play a major role in immunity and autoimmune disease, and insights
into their mechanisms may one day help treat or even prevent such diseases
as arthritis, diabetes, and multiple sclerosis (possibly even AIDS).
Comparative analysis is also used to model human genetic diseases. Given
sequence information, researchers can produce targeted mutations in the
mouse as a rapid and economical route to elucidating gene function. Such
studies continue to be used effectively at Oak Ridge National Laboratory
(ORNL).
DNA
Sequencing
From the beginning of the genome project, DOE's DNA sequencing-technology
program has supported both improvements to established methodologies and
innovative higher-risk strategies. The first major sequencing project,
a test bed for incremental improvements, culminated with elucidation of
the highly complex TCR region (described above) by a team led by Hood.
A novel "directed" sequencing strategy initiated at LBNL in 1993 provides
a potential alternative approach that can include automation as a core
design feature. In this approach, every sequencing template is first mapped
to its original position on a chromosome (resolution, 30 bases). The advantages
of this method include a large reduction in the number of sequencing reactions
needed and in the sequence-assembly steps that follow. To date, this directed
strategy has achieved significant results with simpler, less repetitive
nonhuman sequences, particularly in the NIH-funded Drosophila genome
program. The system also is in use at the Stanford Human Genome Center
and Mercator Genetics, Inc.
The preparation of DNA clones for sequencing involves several biochemical
processing steps that require different solution environments. At the Whitehead
Institute, Trevor Hawkins has improved systems for reversible binding of
DNA molecules to magnetic beads that are compatible with complete robotic
management. The second-generation Sequatron fits on a tabletop with a single
robotic arm moving sample trays between servicing stations. This very compact
system, supported by sophisticated software, may be ideal for laboratories
with limited or costly floor space.
Fluorescent tags are critical components of conventional automated sequencing
approaches. The team of Richard Mathies and Alexander Glazer (University
of California, Berkeley) has made a series of improvements in fluorescence
systems that have decreased DNA input needs and markedly increased the
quality of raw data, thereby supporting longer useful reads of DNA sequence.
Complementary improvements in enzymology have been achieved by the team
of Charles Richardson and Stanley Tabor (Harvard Medical School). Current
widely used procedures for automated DNA sequencing involve cycling between
high and low temperatures. The Harvard researchers used information about
the three-dimensional structure of polymerases (enzymes needed for DNA
replication) and how they function to engineer an improved Taq polymerase.
ThermoSequenase, which is now produced commercially as part of the ThermoSequenase
kit, reduces the amount of expensive sequencing reagents required and supports
popular cycle-sequencing protocols.
The application of higher electrical fields in gel electrophoresis
separation of DNA fragments can increase sequencing speed and efficiency.
Conventional thick gels cannot adequately dissipate the additional heat
produced, however. Two promising routes to "thinness" are ultrathin slab
gels and capillary systems. An ultrathin gel system was developed by Lloyd
Smith (University of Wisconsin, Madison) and licensed for commercial development.
The replacement of gels by pumpable solutions of long polymers is making
capillary array electrophoresis (CAE) potentially practical for DNA sequencing.
The first CAE system for DNA was demonstrated by the team of Barry Karger
(Northeastern University). In 1995, Karger and Norman Dovichi (University
of Alberta, Canada) separately identified CAE conditions under which DNA
sequencing reads could be extended usefully up to the 1000-base range.
Another CAE system, developed by Edward Yeung (Iowa State University),
has been licensed for commercial production (see
box). Mathies has developed a system in which a confocal microscope
displays DNA bands. Application of this system to the sizing of larger
DNA fragments binding multiple fluors allows single-molecule detection.
Replacing the gel-separation step with mass spectroscopy (MS) is another
promising approach for rapid DNA sequencing. MS uses differences in mass-to-charge
ratios to separate ionized atoms or molecules. Early efforts at MS sequencing
were plagued by chemical reactivity during the "launching" phase of matrix-assisted
laser desorption ionization (MALDI). MALDI badly degraded the DNA sample
input. However, the degradation chemistry was elucidated in Smith's laboratory,
leading to improvements. At ORNL, the team of Chung-Hsuan Chen has performed
extensive trials of alternative matrices and has achieved significant improvements
that now support sequence reads up to 100 DNA bases. The system is undergoing
trials for DNA diagnostic applications.
The most revolutionary sequencing technology is being pursued by the
team of Richard Keller and James Jett at LANL. Their goal is to read out
sequence from single DNA molecules, work that builds on LANL's expertise
in flow cytometry. The strand to be sequenced is labeled first with fluors
that distinguish the four DNA subunits and is then suspended in a flow
stream. An exonuclease cleaves the subunits, which flow past an interrogating
laser system that reports the subunits' identities. All system constituents
are operational but limited by the low subunit release rates of commercially
available exonucleases. A current developmental focus is on identifying
more active exonucleases.
Synthetic DNA strands in the 15- to 30-base range (oligomers) play essential
roles in DNA sequencing; in sample-preparation steps for the polymerase
chain reaction, which copies DNA strands millions of times; and in DNA-based
diagnostics. The cost of custom oligomer synthesis once was a limiting
factor in many research projects. A more economical, highly parallel oligomer
synthesis technology was developed by Thomas Brennan at Stanford University.
The sequencing-by-hybridization (SBH) technology provides information
only on short stretches of DNA in a single trial (interrogation), but thousands
of low-cost interrogations can be performed in parallel. SBH is very useful
for rapid classification of short DNAs such as cDNAs, very low cost DNA
resequencing, and detection of DNA sequence differences (polymorphisms)
over short regions. The team of Radomir Crkvenjakov and Radoje Drmanac
invented one format of SBH while in Yugoslavia, made substantial improvements
at Argonne National Laboratory (ANL), and later started Hyseq Inc. to commercialize
these technologies. At ANL, another implementation, SBH on matrices (SHOM)
of gels, holds promise for high-accuracy sequence proofreading and diverse
DNA diagnostics. The ANL team, led by Andrei Mirzabekov, collaborates with
the Englehardt Institute in Moscow, where SHOM was demonstrated initially.
