Mapping the Terrain
One of the central goals of the Human Genome
Project is to produce a detailed "map" of
the human genome. But, just as there are topographic
maps and political maps and highway maps of the
United States, so there are different kinds of genome
maps, the variety of which is suggested in Genomic geography. One type,
a genetic linkage map, is based on careful
analyses of human inheritance patterns. It indicates
for each chromosome the whereabouts of genes or other
"heritable markers," with distances
measured in centimorgans, a measure of recombination
frequency. During the formation of sperm and egg
cells, a process of genetic recombination -- or
"crossing over" -- occurs in which pieces
of genetic material are swapped between paired
chromosomes. This process of chromosomal scrambling
accounts for the differences invariably seen even in
siblings (apart from identical twins). Logically, the
closer two genes are to each other on a single
chromosome, the less likely they are to get split up
during genetic recombination. When they are close
enough that the chances of being separated are only
one in a hundred, they are said to be separated by a
distance of one centimorgan.
The role of human pedigrees now becomes clear. By
studying family trees and tracing the inheritance of
diseases and physical traits, or even unique segments
of DNA identifiable only in the laboratory,
geneticists can begin to pin down the relative
positions of these genetic markers. By the end of
1994, a comprehensive map was available that included
more than 5800 such markers, including genes
implicated in cystic fibrosis, myotonic dystrophy,
Huntington disease, Tay-Sachs disease, several
cancers, and many other maladies. The average gap
between markers was about 0.7 centimorgan.
Other maps are known as physical maps, so
called because the distances between features are
measured not in genetic terms, but in
"real" physical units, typically, numbers
of base pairs. A close analogy can thus be drawn
between physical maps and the road maps familiar to
us all. Indeed, the analogy can be extended further.
Just as small-scale road maps may show only large
cities and indicate distances only between major
features, so a low-resolution physical map includes
only a relative sprinkling of chromosomal landmarks.
A well-known low-resolution physical map, for
example, is the familiar chromosomal map, showing the
distinctive staining patterns that can be seen in the
light microscope. Further, by a process known as in
situ hybridization, specific segments of DNA can
be targeted in intact chromosomes by using
complementary strands synthesized in the laboratory.
These laboratory-made "probes" carry a
fluorescent or radioactive label, which can then be
detected and thus pinpointed on a specific region of
the chromosome. Fishing for
genes shows some results of fluorescence in situ
hybridization (FISH). Of particular interest are
probes known as cDNA (for complementary DNA),
which are synthesized by using molecules of messenger
RNA as templates. These molecules of cDNA thus
hybridize to "expressed" chromosomal
regions -- regions that directly dictate the
synthesis of proteins. However, a physical map that
depended only on in situ hybridization would be a
fairly coarse one. Fluorescent tags on intact
chromosomes cannot be resolved into separate spots
unless they are two to five million base pairs apart.
Fortunately, means are also available to produce
physical maps of much higher resolution -- analogous
to large-scale county maps that show every village
and farm road, and indicate distances at a similar
level of detail. Just such a detailed physical map is
one that emerges from the use of restriction
enzymes -- DNA-cleaving enzymes that serve as
highly selective microscopic scalpels (see Tools of the Trade). A
typical restriction enzyme known as EcoRI, for
example, recognizes the DNA sequence GAATTC and
selectively cuts the double helix at that site. One
use of these handy tools involves cutting up a
selected chromosome into small pieces, then cloning
and ordering the resulting fragments. The cloning,
or copying, process is a product of recombinant DNA
technology, in which the natural reproductive
machinery of a "host" organism -- a
bacterium or a yeast, for example -- replicates a
"parasitic" fragment of human DNA, thus
producing the multiple copies needed for further
study (see Tools of the Trade).
By cloning enough such fragments, each overlapping
the next and together spanning long segments (or even
the entire length) of the chromosome, workers can
eventually produce an ordered library of clones. Each
contiguous block of ordered clones is known as a contig
(a small one is shown in Genomic
geography), and the resulting map is a contig
map. If a gene can be localized to a single fragment
within a contig map, its physical location is thereby
accurately pinned down. Further, these conveniently
sized clones become resources for further studies by
researchers around the world -- as well as the
natural starting points for systematic sequencing
efforts.
Two giant steps: Chromosomes 16 and 19
One of the signal achievements of the DOE genome
effort so far is the successful physical mapping of
chromosomes 16 and 19. The high-resolution chromosome
19 map, constructed at the Lawrence Livermore
National Laboratory, is based on restriction
fragments cloned in cosmids, synthetic cloning
"vectors" modeled after bacteria-infecting
viruses known as bacteriophages. Like a phage, a
cosmid hijacks the cellular machinery of a bacterium
to mass-produce its own genetic material, together
with any "foreign" human DNA that has been
smuggled into it. The foundation of the chromosome 19
map is a large set of cosmid contigs that were
assembled by automated analysis of overlapping but
unordered restriction fragments. These contigs span
an estimated 54 million base pairs, more than 95
percent of the chromosome, excluding the centromere.
Most of the contigs have been mapped by
fluorescence in situ hybridization to visible
chromosomal bands. Further, more than 200 cosmids
have been more accurately ordered along the
chromosome by a high-resolution FISH technique in
which the distances between cosmids are determined
with a resolution of about 50,000 base pairs. This
ordered FISH map, with cosmid reference points
separated by an average of 230,000 base pairs,
provides the essential framework to which other
cosmid contigs can be anchored. Moreover, the EcoRI
restriction sites have been mapped on more than 45
million base pairs of the overall cosmid map. Over
450 genes and genetic markers have also been
localized on this map, of which nearly 300 have been
incorporated into the ordered map. An emerging gene map shows
the locations of the mapped genes. Among these genes
is the one responsible for the most common form of
adult muscular dystrophy (DM), which was identified
in 1992 by an international consortium that included
Livermore scientists. A second important disease gene
(COMP), responsible for a form of dwarfism known as
pseudoachondroplasia, has also been identified. And
yet another gene, one linked to a form of congenital
kidney disease, has been localized to a single contig
spanning one million base pairs, but has not yet been
precisely pinpointed. About 2000 other genes are
likely to be found eventually on chromosome 19.
In a similar effort, the Los
Alamos National Laboratory Center for Human Genome
Studies has completed a highly integrated map of
chromosome 16, a chromosome that contains genes
linked to blood disorders, a second form of kidney
disease, leukemia, and breast and prostate cancers. A
readable display of this integrated map covers a
sheet of paper more than 15 feet long; a portion of
it, much reduced and showing only some of its central
features, is reproduced here as Mapping chromosome 16. The
framework for the Los Alamos effort is yet another
kind of map, a "cytogenetic breakpoint map"
based on 78 lines of cultured cells, each a hybrid
that contains mouse chromosomes and a fragment of
human chromosome 16. Natural breakpoints in
chromosome 16 are thus identified, leading to a
breakpoint map that divides the chromosome into
segments whose lengths average 1.1 million base
pairs. Anchored to this framework are a
low-resolution contig map based on YAC clones and a
high-resolution contig map based largely on cosmids
(for more on YACs, yeast artificial chromosomes, see Tools of the Trade). The
low-resolution map, comprising 700 YACs from a
library constructed by the Centre d'Etude du
Polymorphisme Humain (CEPH), provides practically
complete coverage of the chromosome, except the
highly repetitive DNA in the centromere region. The
high-resolution map comprises some 4000 cosmid
clones, assembled into about 500 contigs covering 60
percent of the chromosome. In addition, it includes
250 smaller YAC clones that have been merged with the
cosmid contig map. The cosmid contig map is an
especially important step forward, since it is a
"sequence-ready" map. It is based on
bacterial clones that are ideal substrates for DNA
sequencing, and further, these clones have been
restriction mapped to allow identification of a
minimum set of overlapping clones for a large-scale
sequencing effort.
The high- and low-resolution maps have been tied
together by sequence-tagged sites (STSs), short but
unique stretches of DNA sequence. They have also been
integrated into the breakpoint map, and with genetic
maps developed at the Adelaide Children's Hospital
and by CEPH. The integrated map also includes a
transcription map of 1000 sequenced exons
(expressed fragments of genes) and more than 600
other markers developed at other laboratories around
the world.
Getting down to details: Sequencing the genome
Ultimately, though, these physical maps and the
clones they point to are mere stepping stones to the
most visible goal of the genome project, the string
of three billion characters -- A's, T's, C's, and G's
-- representing the sequence of base pairs that
defines our species. Included, of course, would be
the sequence for every gene, as well as the sequences
for stretches of DNA whose functions we don't yet
know (but which may be involved in such
little-understood processes as orchestrating gene
expression in different parts of our bodies, at
different times of our lives). Should anyone
undertake to print it all out, the result would fill
several hundred volumes the size of a big-city phone
book.
Only the barest start has been made in taking this
dramatic step in the Human Genome Project. Several
hundred million base pairs have been sequenced and
archived in databases, but the great majority of
these are from short "sequence tags" on
cloned fragments. Only about 30 million base pairs of
human DNA (roughly one percent of the total) have
been sequenced in longer stretches, the longest being
about 685,000 base pairs long. Even more daunting is
the realization that we will eventually need to
sequence many parts of the genome many times, thus to
reveal differences that indicate various forms of the
same gene.
Hence, as with so many human enterprises, the
challenge of sequencing the genome is largely one of
doing the job cheaper and faster. At the beginning of
the project, the cost of sequencing a single base
pair was between $2 and $10, and one researcher could
produce between 20,000 and 50,000 base pairs of
continuous, accurate sequence in a year. Sequencing
the genome by the year 2005 would therefore likely
cost $10-20 billion and require a dedicated cadre of
at least 5000 workers. Clearly, a major effort in
technology development was called for -- an effort
that would drive the cost well below $1 per base pair
and that would allow automation of the sequencing
process. From the beginning, therefore, the DOE has
emphasized programs to pave the way for expeditious
and economical sequencing efforts -- programs to
develop new technologies, including new cloning
vectors, and to establish suitable resources for
sequencing, including clone libraries and libraries
of expressed sequences.
Efforts to develop new cloning vectors have been
especially productive. YACs remain a classic tool for
cloning large fragments of human DNA, but they are
not perfect. Some regions of the genome, for example,
resist cloning in YACs, and others are prone to
rearrangement. New vectors such as bacterial
artificial chromosomes (BACs), P1 phages, and
P1-derived artificial cloning systems (PACs) have
thus been devised to address these problems. These
new approaches are critical for ensuring that the
entire genome can be faithfully represented in clone
libraries, without the danger of deletions,
rearrangements, or spurious insertions.
Marked progress is also evident in the development
of sequencing technologies, though all of those in
widespread current use are still based on methods
developed in 1977 by Allan Maxam and Walter Gilbert
and by Frederick Sanger and his coworkers (see Tools of the Trade). Both of
these methods rely on gel-based electrophoresis
systems to separate DNA fragments, and recent
advances in commercial systems include increasing the
number of gel lanes, decreasing run times, and
enhancing the accuracy of base identification. As a
result of such improvements, a standard sequencing
machine can now turn out raw, unverified sequences of
50,000 to 75,000 bases per day.
Equally important to the sequencing goals of the
genome project is a rational system for organizing
and distributing the material to be sequenced. The
DOE's commitment to such resources dates back to
1984, when it organized the National Laboratory Gene
Library Project. Based on cell- and
chromosome-sorting technologies developed at
Livermore and Los Alamos, libraries of clones were
established for each of the human chromosomes, and
the individual clones are widely available for
mapping and for isolating genes. These clones were
invaluable in such notable "gene hunts" as
the successful searches for the cystic fibrosis and
Huntington disease genes. More recently, as more
efficient vectors have become available, complete
human DNA libraries have been established using BACs,
PACs, and YACs.
Another critical resource is being assembled in an
effort known as I.M.A.G.E. (Integrated Molecular
Analysis of Genomes and their Expression), cofounded
by the Livermore
Human Genome Center. The aim is a master set of
mapped and sequenced human cDNA, representing the
expressed parts of the human genome. By early 1996,
I.M.A.G.E. had distributed over 250,000 partial and
complete cDNA clones, most of them with one or both
ends sequenced to provide unique identifiers. These
identifiers, expressed sequence tags (ESTs),
are usually 300-500 base pairs each. Twenty-five
hundred genes have also been newly mapped as part of
this coordinated effort.
Shotguns and transposons
Such advances as these, in both technology
development and the assembly of resource libraries,
have brought much nearer the day when
"production sequencing" can begin. A great
deal of variety remains, however, in the approaches
available to sequencing the human genome, and it is
not yet clear which will prove the most efficient and
most cost-effective way to read long stretches of DNA
over the next decade. One of the available choices,
for example, is between "shotgun" and
"directed" strategies. Another is the
degree of redundancy -- that is, how many times must
a given strand be sequenced to ensure acceptable
confidence in the result?
Shotgun sequencing derives its name from the
randomly generated DNA fragments that are the objects
of scrutiny. Many copies of a single large clone are
broken into pieces of perhaps 1500 base pairs, either
by restriction enzymes or by physical shearing. Each
fragment is then separately cloned, and a convenient
portion of it sequenced. A computational assembly
process then compares the terminal sequences of the
many fragments and, by finding overlaps that indicate
neighboring fragments, constructs an ordered library
for the parent clone. The members of this ordered
library can then be sequenced from end to end to
yield a complete sequence for the parent. The
statistics involved in taking this approach require
that many copies of the original clone be randomly
fragmented, if no gaps are to be tolerated in the
final sequence. A benefit is that the final sequence
is highly reliable; the main disadvantage is that the
same sequence must be done many times (in the many
overlapping fragments). Nevertheless, shotgun
sequencing has been the primary means for generating
most of the genomic sequence data in public DNA
databases. This includes the longest contiguous
fragment of sequenced human DNA, from the human
T-cell receptor beta region, of about 685,000 base
pairs -- a product of DOE-supported work at the
University of Washington.
The shotgun strategy is also being used at the
Genome Therapeutics Corporation and The Institute for
Genomic Research (TIGR), as part of the DOE-supported
Microbial Genome Initiative. Genome Therapeutics has
sequenced 1.8 million base pairs of Methanobacterium
thermoautotrophicum, a bacterium important in
energy production and bioremediation, and TIGR has
successfully sequenced the complete genomes of three
free-living bacteria, Haemophilus influenzae
(1,830,137 base pairs; an effort supported mostly by
private funds), Mycoplasma genitalium (580,070
base pairs), and Methanococcus jannaschii
(1,739,933 base pairs).
The alternative to shotgun sequencing is a
directed approach, in which one seeks to sequence the
target clone from end to end with a minimum of
duplication. The essence of this approach is embodied
in a technique known as primer walking.
Starting at one end of a single large fragment, one
replicates a stretch of DNA -- say, 400 base pairs
long -- that can be sequenced in one run. With the
sequence for this first segment in hand, the next
stretch of DNA, just overlapping the first, is then
tackled in the same way. In principle, one can thus
"walk" the entire length of the original
clone. Unfortunately, this conceptually simple
approach has been historically beset with
disadvantages, mainly the expense and inconvenience
of custom-synthesizing a primer as the necessary
starting point for each sequencing step. The widely
automated Sanger sequencing method involves a DNA
replication step that must be "primed" by a
DNA fragment that is complementary to 15 to 20 base
pairs of the strand to be sequenced (see Tools of the Trade). Until
recently, making these primers was an expensive and
time-consuming business, but recent innovations have
made primer walking, and similar directed strategies,
more and more economically feasible.
One way to deal with the primer bottleneck, for
example, is to use sets of very short fragments to
prime the next sequencing step. As an illustration,
the four nucleotides (A, T, C, and G) can be ordered
in more than 68 billion ways to create an 18-base
primer, an imposing set of possibilities. But it is
eminently practical to create a library of the 4096
possible 6-base primers. Three of these
"6-mers" can be matched to the end of the
fragment to be sequenced, thus serving as an 18-base
primer. This modular primer technology, developed at
the Brookhaven National
Laboratory, is currently being applied to Borrelia
burgdorferi, the organism that causes Lyme
disease; a 34,000-base-pair fragment has already been
sequenced.
Another directed approach uses a naturally
occurring genetic element called a transposon,
which insinuates itself more or less randomly in
longer DNA strands. This predilection for random
insertion and the fact that the transposon's DNA
sequence is well known are the keys to the sequencing
strategy depicted schematically in Taking a directed approach.
The largest clones are broken into smaller subclones
(each of about 3000 base pairs), which then become
the targets of the transposons. Multiple copies of
each subclone are exposed to the transposons, and
reaction conditions are controlled to yield, on
average, a single insertion in each 3000-base-pair
strand. The individual strands are then analyzed to
yield, for each, the approximate position of the
inserted transposon. By mapping these positions, a
"minimum tiling path" can be determined for
each subclone -- that is, a set of strands can be
identified whose transposon insertions are roughly
300 base pairs apart. In this set of strands, the
region around each transposon is then sequenced,
using the inserted transposons as starting points.
The known transposon sequence allows a single primer
to be used for sequencing the full set of overlapping
regions.
At the Lawrence
Berkeley National Laboratory, this technique has
been used to sequence over 1.5 million base pairs of
DNA on human chromosomes 5 and 20, as well as over
three million base pairs from the fruit fly Drosophila
melanogaster. On chromosome 5, interest focuses
on a region of three million base pairs that is rich
in growth factor and receptor genes; whereas, on
chromosome 20, Berkeley researchers are interested in
a region of about two million base pairs that is
implicated in 15 to 20 percent of all primary breast
carcinomas. As an example of the kind of output these
efforts produce, Sequence
data: The final product shows a stretch of
sequence data from chromosome 5.
Researchers supported by the DOE at the University of Utah
are also pursuing the use of directed sequencing. In
addition, they have developed a methodology for
"multiplex" DNA sequencing, which offers a
way of increasing throughput with either shotgun or
directed approaches. By attaching a unique
identifying sequence to each sequencing sample in a
mixture of, say, 50 such samples, the entire mixture
can be analyzed in a single electrophoresis lane. The
50 samples can be resolved sequentially by probing,
first, for bands containing the first identifier,
then for bands containing the second, and so forth.
In a similar way, multiplexing can also be used for
mapping. The Utah group is now able to map almost
5000 transposons in a single experiment, and they are
using multiplexing in concert with a directed
sequencing strategy to sequence the 1.8 million base
pairs of the thermophilic microbe Pyrococcus
furiosus and two important regions of human
chromosome 17.
The completed physical maps of chromosomes 16 and
19, with their extensive coverage in many different
kinds of cloning vectors, are especially ripe for
large-scale sequencing. Los Alamos scientists have
therefore begun sequencing chromosome 16, focusing
special effort on locating the estimated 3000
expressed genes on that chromosome and using those
sites as starting points for directed genomic
sequencing. A region of 60,000 base pairs has already
been sequenced around the adult polycystic kidney
gene, and good starts have been made in mapping other
genes. Interestingly, even random sequencing has led
to the identification of gene DNA in over 15 percent
of the samples, confirming the apparent high density
of genes on this chromosome. Between chromosome 16
and the short arm of chromosome 5, another Los Alamos
target, the genome center there has produced almost
two million base pairs of human DNA sequence.
A parallel effort is under way at Livermore on
chromosome 19 and other targeted genomic regions.
Using a shotgun approach, researchers there have
completed over 1.3 million bases of genomic sequence.
Initially, they are attacking two major regions of
chromosome 19: one of about two million base pairs,
containing several genes involved in DNA repair and
replication, and another of approximately one million
base pairs, containing a kidney disease gene. The
Livermore scientists are making use of the I.M.A.G.E.
cDNA resource to sequence the cDNA from these
regions, along with the associated segments of the
genome. In addition, Livermore scientists have
targeted DNA repair gene regions throughout the
genome and, in many cases, have done comparative
sequencing of these genes in other species,
especially the mouse. Such comparative sequencing has
identified conserved sequence elements that might act
as regulatory regions for these genes and has also
assisted in the identification of gene function (see The Mighty Mouse).
How good is good enough?
The goal of most sequencing to date has been to
guarantee an error rate below 1 in 10,000, sometimes
even 1 in 100,000. However, the difference between
one human being and another is more like one base
pair in five hundred, so most researchers now agree
that one error in a thousand is a more reasonable
standard. To assure a higher level of confidence, and
perhaps to uncover important individual differences,
the most biologically or medically important regions
would still be sequenced more exhaustively, but using
this lowered standard would greatly reduce the cost
of acquiring sequence data for the bulk of human DNA.
With this philosophy in mind, Los Alamos
scientists have begun a project to determine the cost
and throughput of a low-redundancy sequencing
strategy known as sample sequencing (SASE, or
"sassy"). Clones are selected from the
high-resolution Los Alamos cosmid map, then
physically broken into 3000-base-pair subclones --
much as in other sequencing approaches. In contrast
to, say, shotgun sequencing, though, only a small
random set of the subclones is then selected for
sequencing. Sequence fragments already known -- end
sequences, sequence-tagged sites, and so forth -- are
used as the starting points. The result is sequence
coverage for about 70 percent of the original cosmid
clone, enough to allow identification of genes and
ESTs, thus pinpointing the most critical targets for
later, more thorough sequencing efforts. Further, the
SASE-derived sequences provide enough information for
researchers elsewhere to pursue just such
comprehensive efforts, using whole genomic DNA. In
addition, the cost of SASE sequencing is only
one-tenth the cost of obtaining a complete sequence,
and a genomic region can be "sampled" ten
times as fast.
As the first major target of SASE analysis, Los
Alamos scientists chose a cosmid contig of four
million base pairs at the end (the telomere)
of the short arm of chromosome 16. By early 1996,
over 1.4 million base pairs had been sequenced, and a
gene, EST, or suspected coding region had been
located on every cosmid sampled.
In addition, Los Alamos is building on the SASE
effort by using SASE sequence data as the basis for
an efficient primer walking strategy for detailed
genomic sequencing. The first application of this
strategy, to a telomeric region on the long arm of
chromosome 7, proved to be as efficient as typical
shotgun sequencing, but it required only two- to
threefold redundancy to produce a complete sequence,
in contrast to the seven- to tenfold redundancy
required in shotgun approaches. The resulting
230,000-base-pair sequence is the second-longest
stretch of contiguous human DNA sequence ever
produced.
In a sense, though, even a complete genome
sequence -- the ultimate physical map -- is only a
start in understanding the human genome. The deepest
mystery is how the potential of 100,000 genes is
regulated and controlled, how blood cells and brain
cells are able to perform their very different
functions with the same genetic program, and how
these and countless other cell types arise in the
first place from an single undifferentiated egg cell.
A first step toward solving these subtle mysteries,
though, is a more complete physical picture of the
master molecules that lie at the heart of it all.
SIDEBAR: Tools of the
Trade
SIDEBAR: The Mighty Mouse
To Know
Ourselves
was prepared at the request of the U.S. Department of Energy,
Office of Health and Environmental Research, as an
overview of the Human
Genome Project.