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2
C HAPTE R
Human Genome Project and its
Ethical Issues
2.1. INTRODUCTION
The Human Genome Project (HGP) is an 13-year
effort, which formally initiated in October 1990.
The first idea of Human Genome Project came
from the discussions held during scientific
meetings, which were organized by the US
department of energy and other scientific
organizations between 1984 and 1986. The project
was planned, spanning a period of 15 years, but
rapid technological advances accelerated its
completion within 13 years, i.e., in 2003. The three
billion US dollars funds were earmarked for the
sequencing of more than two meters length of
human DNA. The goal of the project was to
determine the complete sequence of the three
billion (3 × 109) DNA subunits (bases), identify
all human genes, and make them accessible for
further biological study. As a part of the HGP,
parallel sequencing was done for selected model
organisms, such as the bacterium E. coli to help
develop the technology and interpret human gene
function. The Department of Energy’s ‘Human
Genome Program (HGP)’ and the National
Institutes of Health’s ‘National Human Genome
Research Institute (NHGRI)’ together sponsored
the US Human Genome Project. Ari Patrinos, head
of the Office of Biological and Environmental
Research, directed the Department of Energy’s
‘Human Genome Program’ research. Francis
Collins directed the National Institutes of Health’s,
National Human Genome Research Institute
efforts.
The Corporate Genome Project was initiated
rather late by Celera Genomics, a company
founded by a former NIH scientist, Craig Venter,
and funded by Perkin-Elmer, a large
instrumentation manufacturer that makes and sells
instruments to the government and to the private
sector as well.
The Human Genome Project (HGP) and the
Corporate Genome Project (CGP) are two very
distinctly different entities having different cultures
and attitudes. The focus has been around the
privacy issue. Celera wanted to retain some of the
information or control over the information that it
was going to publish about the human genome,
because, in fact, its business model was dependent
on that fact.
From the inception of this project, due to the
huge budget in sequencing of human DNA, many
laboratories around the United States received
Human Genome Project and its Ethical Issues 17
determine, among other things, how the organism
looks, how well its body metabolizes food or fights
infection, and sometimes even how it behaves. The
human genome is made up of DNA (which has
four different chemical building blocks).
DNA is made up of four similar chemicals
(called bases and abbreviated A, T, C and G) that
are repeated millions or billions of times
throughout a genome. The human genome, for
example, has 3 billion pairs of bases.
In DNA, the particular order of As, Ts, Cs and
Gs is extremely important. The order underlies
the life’s diversity, even dictating whether an
organism is human or another species, such as
yeast, rice, or fruit fly, all of which have their own
genomes and are themselves the focus of genome
projects. Since all organisms are related through
similarities in DNA sequences, insights gained
from nonhuman genomes often lead to new
knowledge about human biology. To get an idea
of the size of the human genome present in each
of our cells, consider the following analogy: If the
DNA sequence of the human genome were
compiled in books, the equivalent of 200 volumes
the size of a telephone book (at 1000 pages each)
would be needed to hold it all (Fig. 2.1).
Storing all this information is a great challenge
for computer experts known as bioinformatics
specialists. One million bases (called a mega base
and abbreviated Mb) of DNA sequence data is
roughly equivalent to 1 megabyte of computer data
storage space. Since the human genome is 3 billion
base pairs long, 3 gigabytes of computer data
storage space is needed to store the entire genome.
This includes only nucleotide sequence data, and
does not include data annotations and other
information that can be associated with sequence
data.
As time goes on, more annotations will be
entered as a result of laboratory findings, literature
searches, data analyses, personal communications,
automated data-analysis programs, and auto
annotators. These annotations associated with the
sequence data are likely to dwarf the amount of
Fig. 2.1. Compiling the DNA sequence from the
human genome into books would require
200 volumes, each the size of the 1,000 page
Bangalore telephone book.
storage space actually taken up by the initial
3 billion nucleotide sequence. Of course, that’s not
much of a surprise because the sequence is merely
a starting point for a much deeper biological
understanding.
Human beings are also similar to other living
cells in their basic cell characteristics.
Cells: These are the fundamental working
units of every living system. All the instructions
Human Genome Project and its Ethical Issues 19
Fig. 2.2. Human genome and nature of DNA.
blocks, G and C. In contrast, the gene-poor
“deserts” are rich in the DNA building blocks, A
and T. GC- and AT-rich regions can usually be seen
through a microscope as light and dark bands on
chromosomes.
Genes appear to be concentrated in random
areas along the genome, with vast expanses of noncoding DNA in between.
Stretches of upto 30,000 C and G bases
repeating over and over often occur adjacent to
gene-rich areas, forming a barrier between the
genes and the “junk DNA”. These CpG islands
are believed to help regulate gene activity.
Chromosome 1 has most of the genes (2968), and
the Y chromosome has the fewest (231).
Scientists have identified about 1.4 million
locations where single-base DNA differences i.e.,
single nucleoride polymorphisms (SNPs) occur
in humans. This information promises to
revolutionize the processes of finding
chromosomal locations for disease-associated
sequences and tracing human history.
2.4. GENOME SEQUENCING
Genome sequencing is the term used to describe
the laboratory process of reading the order of the
four letters of the genetic alphabet (A, C, G, T)
along a strand of DNA. The steps involved in such
efforts are as follows:
1. Selection of suitable sample materials.
2. Isolation of DNA from the cells, and
preparation of large samples of high quality
DNA from these cells.
3. Cutting the purified DNA at random sites into
a manageable size, overlapping pieces of the
DNA sample.
4. Insertion of these DNA pieces into packages
for the production of unlimited copies of such
selected DNA.
5. Recording the order of bases for each DNA
sample piece by using DNA sequencing
techniques.
6. Determination of the overlap of each piece,
and assembling the sequences to give the final
genome of the human.
20 Bioethics and Biosafety
While following the above approaches, it is
necessary to make appropriate sample populations
based on the distribution of humans. A primary
goal of the Human Genome Project is to generate
detailed maps of the human genome. These maps
will aid in determining the location of genes within
the human genome. More specifically, they will
assign genes to their chromosomes. Two types of
maps are being generated genetic linkage maps
and physical maps.
Genetic linkage maps determine the relative
arrangement and approximate distances between
genes and markers on the chromosomes and
physical maps specify the physical location (in base
pairs) and distance between genes or DNA
fragments with unknown functions that are mapped
to specific regions of the chromosomes.
Maps have different levels of resolution,
ranging from low to high. The degree of resolution
that is appropriate depends on whether, for
example, a large fragment of DNA is to be studied
or a more detailed picture of a small DNA region
is needed. A human genomic library consists of
random DNA fragments, and is used to establish
sets of ordered, overlapping cloned DNA
fragments or contigs for each chromosome of the
genome, In other words, these are high-resolution
maps.
After mapping is complete, the DNA must be
sequenced to determine the order of all the
nucleotide bases of the chromosomes, and the
genes in the DNA sequence must be identified. In
all phases of the project, a major focus has been on
developing instrumentation to increase the speed
of data collection and analysis. New, automated
technologies are significantly increasing the speed
and accuracy of DNA sequencing, while
decreasing the cost. Software and database systems
manage the data generated from mapping and
sequencing projects. Database management
systems store and aid in distributing genomic
information (Fig. 2.3).
Genetic linkage maps show the order and
genetic distance between pairs of linked genes, that
is, genes on the same chromosome that determine
variable phenotypic traits (the difference between
genetic distance and physical distance is explained
below). Genetic linkage maps enable the
geneticists to follow the inheritance of specific
traits (that is, genes) as they are passed from
generation to generation within the families.
Linkage maps also determine the arrangement
of genes or markers with unknown functions on
the chromosomes. They show the order of linked
genes and pairwise distances between their loci.
During meiosis, as the haploid egg and sperm cells
form, homologous chromosomes (maternally and
paternally derived) line up, and DNA segments
can be exchanged between the homologs. The new
combinations of alleles result from this process of
homologous recombination. During meiosis, each
human chromosome pair is involved, on an
average, in 1.5 crossover events. The likelihood
of crossing over increases as the distance between
the two loci increases. Crossing over between two
genes or markers on the same chromosome can
sometimes occur if there is enough distance
between them. If two genes are very close, they
are “linked” and recombination is unlikely to occur
between them. Thus, the frequency of
recombination is a quantitative index of the linear
distance between two genes on a genetic linkage
map. Distances are measured in centimorgans
(cM), named after the famous geneticist, Thomas
Hunt Morgan. If genes (for example, A and B) are
separated by recombination 1% of the time, that
is, if one out of 100 products of meiosis is
recombinant, they are 1 cM apart. A genetic
distance of 1 cM represents a physical distance of
approximately one million base pairs (1 Mb).
Genetic maps are very powerful. An inherited
disease gene can be located on the map if a second
gene or DNA reference marker is also inherited in
individuals with the disease, but is not found in
individuals who do not have that disease. Exact
chromosomal locations have already been found
for many disease genes, including fragile X
syndrome, cystic fibrosis and Buntington’s disease.
Human Genome Project and its Ethical Issues 21
Fig. 2.3. Process of determination of DNA sequence from human chromosome.
22 Bioethics and Biosafety
Many inherited diseases are caused by single
genes, and thus can be studied by genetic linkage
analysis. Almost 5,000 genetic disorders have been
studied in this way. These maps, however, do not
relate directly to the physical structure of DNA,
and the gene of interest cannot be isolated on the
basis of information from genetic linkage maps
alone or human genome mapping. Linkage
analysis involves the study of family members
carrying a particular trait for an inherited disorder.
Often, several generations of one family are studied
to obtain enough information with which to infer
linkage. Some family members must express the
trait (gene) or genetic disorder, and the trait must
vary among individuals (that is, there must be
different alleles or forms of the gene). Analysis
also requires that there be individuals who are
heterozygous for DNA reference markers or who
have a second gene linked to the gene in question.
Heterozygous family members (members carrying
two different forms of the trait or gene—one
dominant and one recessive allele) enable
geneticists to determine which chromosome of the
homologous pair carries the allele for the genetic
disorder, and whether it is passed on to the
offspring.
The physical location of the DNA marker on
a chromosome can then be found by using the
marker sequence as a DNA probe. Polymorphic
DNA markers serve as reference points or
landmarks to help find a region of DNA that
contains the gene of interest. If a gene is found
between two DNA markers, the DNA region can
be isolated for further study.
An early goal of the investigators of Human
Genome Project was to generate linkage maps with
polymorphic DNA markers, spaced 2 to 5 cM
along each chromosome. This goal was reached
in 1995. Such a map helps scientists to find genes
of interest relative to about 1,500 markers within
the genome, Once linkage maps have some 3,300
polymorphic DNA markers, each separated by only
1 cM, gene hunting will be much easier. Thus, for
polymorphic DNA markers to be valuable, their
linkage with a gene must be established, and their
physical locations must be identified through the
use of probes. Several large scientific groups
working on the human genome are identifying
markers to generate comprehensive genetic maps.
2.5. PHYSICAL MAPS
The physical maps specify the exact physical
location (in base pairs) and distance between genes
or markers, or unknown DNA or genes. These
maps provide information about the physical
organization of the DNA; examples are the
location of restriction enzyme sites and the order
of restriction fragments of chromosomes. An entire
genome can be studied using a library of genomic
DNA. These clones are uncharacterized, random
fragments and are not placed in order, as they
would be on the chromosome.
As the human genome is very large, large
DNA fragments must be cloned into vectors to
maintain manageable number of clones in the
library. Yeast artificial chromosomes (YACs) are
being used as cloning vectors for the human
genome, since a DNA can be up to one million
base pairs in length. Human DNA is attached to
the yeast DNA and transferred into yeast host cells
for replication. Only a small portion of the yeast’s
total DNA, i.e., origin of replication, telomere, and
centromere is required for replication, so most of
the YAC DNA is the foreign DNA.
The average insert used in YAC libraries is
200,000 0 400,000 base pairs in length. This range
is 10 times larger than inserts used in other
libraries, such as for bacteriophage and cosmids,
where up to 20,000 to 40,000 base pairs,
respectively, can be cloned. The human genome
can be represented by 7,500 YAC clones, and is
maintained and amplified in yeast host cells. YACs
and their inserts are cut into smaller fragments and
recloned or subcloned (for example, into cosmids),
so that a detailed map of a YAC clone is obtained.
YAC clones are screened by PCR to isolate
specific genes of interest. DNA inserts are also
Human Genome Project and its Ethical Issues 23
analyzed by obtaining restriction maps, identifying
polymorphic markers, and/or DNA sequencing.
However, without an ordered physical map, i.e.,
one that refers to actual physical distances in base
pairs between landmarks, the location of particular
clones cannot be identified.
Another method called fluorescence in situ
hybridization (FISH) of probes to metaphase
chromosomes provides information for
constructing low-resolution chromosomal maps.
Chromosomal maps are actual physical maps
because distances are measured in base pairs.
Metaphase chromosomes are spread out on a
microscope slide, and a solution containing a
fluorescent-tagged DNA probe is added. Under the
appropriate conditions, the probe hybridizes to its
DNA complement on the chromosome and is
detected with a fluorescent microscope (Figs. 2.4
& 2.5). The relative orientation of genes and DNA
fragments can be assigned to specific
chromosomes, and the gaps between mapped
Fig. 2.4. A microscopic preparation (metaphase
squash before a karyotype is made) of human
chromosomes showing the differences in size and
banding patterns of the chromosomes.
Fig. 2.5. Fluorescence of chromosome position by
probes in fluorescence in situ hybridization.
cosmids can be bridged. Chromosomal mapping
is used to locate genetic markers that are associated
with observable traits.
Another type of physical map is the cDNA
map, which localizes coding regions (for example
exons) to specific chromosome regions or bands.
The cDNA molecules are synthesized from an
mRNA template. The DNA map is probably one
of the most important types of map, since it can
identify the chromosomal location of specific
genes, whether their functions are known or not.
Researchers searching for a specific disease
causing gene can use cDNA maps to help locate it
after having established a general location by
genetic linkage methods.
High-resolution physical maps can be
generated by a method that is sometimes called
bottom-up mapping. The chromosome is cut into
small overlapping fragments, each of which is
cloned and the order determined. These fragments
form continuous DNA blocks called contigs. The
bottom-up method generates a detailed map called
a ‘contig’ map. A library of clones ranging from
10,000 base pairs to 1 Mb is used for mapping.
Each clone can be localized to specific regions
within chromosomal bands. This “linked” library
of overlapping clones comprises a chromosomal
segment.
The production of human contig maps requires
several steps. First, a library must be made that
24 Bioethics and Biosafety
represents the human genome—either the entire
genome or a segment—in cloned DNA fragments.
The DNA fragments within each clone must
overlap other fragments. Overlap is accomplished
by cutting the DNA with a specific restriction
enzyme. If every restriction site on the DNA were
cut, fragments would not overlap. Therefore,
enzyme digestion is conducted in such a way that
only a particular DNA restriction site is cut. This
partial digestion randomly leaves many sites uncut,
so that overlapping DNA fragments are produced
and the order along the chromosomes can be
determined.
The order of the clones or contigs can be
determined by identifying the overlaps in the DNA
fragments. Overlap can be detected when some of
the DNA bands are the same i.e., two clones have
bands in common. This method of assembling pairs
of clones into contigs is difficult and timeconsuming.
Automation and sophisticated computer
algorithms may increase the efficiency. Different
approaches may be used to fill in the gaps that are
likely to be present even after researchers generate
detailed physical maps. For example, microdissection, which is used to physically cut a piece
of DNA from a specific region of a chromosome.
This chromosomal piece can be cut into smaller
fragments by restriction enzymes, cloned, mapped,
and sequenced by standard methods.
An alternate method is “chromosome
walking”, in which a small region at the end of
the DNA fragment is used as a probe to screen the
library for the adjacent clone. A DNA piece at the
end of this second cloned fragment is used as a
next probe. This process continues until a complete
physical map has been obtained. Since the human
genome is divided into chromosomes, chromosome
specific libraries can be constructed so that each
chromosome has a contig map.
Mapping is simplified if each chromosome is
separated from the others before being cut by
restriction enzymes and cloned to make libraries.
Twenty-four libraries are required: 22 autosomal
libraries, and one each for the X and Y
chromosomes. The several types of maps range
from coarse to fine resolution.
The map with the lowest resolution is the
genetic map, which measures the frequency of
recombination between linked markers (which can
be genes or noncoding DNA). The next level of
resolution is the restriction map, on which DNA
restriction fragments ranging from 1 to 2 Mb are
separated and mapped. The next higher level of
resolution is achieved by placing in order 400,000
to 1,000,000 base pair fragments of overlapping
clones from libraries of YAC clones. These clones
are then further subcloned (with insert sizes of
20,000 to 40,000 base pairs) into other vectors to
produce contig maps. Finally, the DNA base
sequence map having the finest resolution is
determined.
Sequence-Tagged Sites
In the sequencing approaches, Human Genome
Project requires that the collected genome
information be shared. A major problem is that
investigators from different laboratories use a
variety of methods for generating and mapping
DNA fragments, thus making correlations difficult
when data from different laboratories are
compared. Therefore, to solve this problem,
universal reference system has been developed.
Unique regions of 200 to 500 base pairs of partially
sequenced DNA are used to identify clones,
contigs, and long stretches of DNA. These
sequence-tagged sites (STSs) are standard markers
that are used for physical mapping. An STS can
also can be a region of cDNA i.e., an exp sequence
called an expressed-sequence tag (EST). ESTs are
used to represent landmarks along the map, thus
helping to identify the regions where pairs of
clones overlap.
These special sequences constitute a “universal
mapping language”, enabling everyone to refer to
a specific region of the genome by the same name,
and enabling investigators to share information and
26 Bioethics and Biosafety
humans, especially in proteins involved in
development and immunity.
The human genome has a much greater portion
(50%) of repeat sequences than the mustard weed
(11%), the worm (7%) and the fly (3%).
Although humans appear to have stopped
accumulating repeated DNA over 50 million years
ago, there seems to be no such decline in rodents.
This may account for some of the fundamental differences between hominids and rodents, though gene
estimates are similar in these species. Scientists
have proposed many theories to explain
evolutionary contrasts between humans and other
organisms, including those of life span, litter sizes,
inbreeding, and genetic drift.
Variations and Mutations
US Human Genome Project Research Goals
The completion of the human DNA sequence in
the spring of 2003 coincided with the 50th
anniversary of Watson and Crick’s description of
the fundamental structure of DNA. The analytical
power arising from the reference DNA sequences
of entire genomes and other genomics resources
has jump-started, what some call the “biology
century”.
The Human Genome Project was marked by
accelerated progress. In June, 2000, the rough draft
of the human genome was completed a year before
its schedule time. In February 2001, special issues
of Science and Nature contained the working draft
sequence and analyses were published.
The project’s first 5-year plan, intended to
guide research in financial years 1990-1995, was
revised in 1993 due to unexpected progress, and
the second plan outlined goals through the FY,
1998. The third and final plan (Science, 23 October
1998) was developed during a series of DOE and
NIH workshops. Some 18 countries have
participated in the worldwide effort, with
significant contributions from the Sanger Center
in the United Kingdom, and research centers in
Germany, France and Japan.
Difference Between Draft Sequence and
Finished Sequence
To generate the high-quality reference sequence,
completed in April 2003, an additional sequencing
was done to close the gaps and reduce the
ambiguities. Further, only a single error was
allowed for every 10,000 bases, the agreed-upon
standard for the HGP. Investigators believe that a
high-quality sequence is critical for recognizing
regulatory components of genes that are very
important in understanding human biology and
disorders such as heart disease, cancer, and
diabetes. The genomes have been sequenced
completely as shown in the Table 2.2.
The small genomes of several viruses and
bacteria, and the much larger genomes of three
higher organisms have been completely sequenced.
They are bakers’ or brewers’ yeast (Saccharomyces
cerevisiae), the roundworm (Caenorhabditis
elegans) and the fruit fly (Drosophila
melanogaster). In October, 2001, the draft
sequence of the pufferfish Fugu rubripes, the first
vertebrate after the human, was completed, and
scientists finished the first genetic sequence of a
plant weed Arabidopsis thaliana, in December
2000. Many more genomes have been sequenced
since then.
Human Genome project is also called Human
Genome Initiative Scientific Research Effort to
analyze the DNA of humans and of several lower
organisms. The project began in the United States
in 1990 under the sponsorship of the US
Department of Energy and the National Institutes
of Health. Projects undertaken concurrently in
Japan, the United Kingdom, Italy, France, and
Russia are coordinated with the American effort
through the Human Genome Organization.
The ultimate goal of the project is to identify
the chromosomal location of every human gene,
and to determine the precise chemical structure of
each gene in order to elucidate its function in health
and disease. The information gathered is expected
to serve as the basic reference for research in
human biology and medicine in the 21st century,
Human Genome Project and its Ethical Issues 27
Table 2.2. The list of organisms whose genome sequence is completed.
Group
Virus
Prokaryotes
Eukaryotes
Organism
MS2
SV40
φX174
M13
λ
Herpes simplex
T2, T4, T6
Smallpox
Methanococcus jannaschii
E. coli
Borrelia burgdorferi
Saccharomyces cerevisiae
Caenorhabditis elegans
Arabidopsis thaliana
Drosophila melanogaster
Homo sapiens
Zea mays
Fugu rubripes
Amphiuma means
and to provide fundamental insights into the
genetic basis of human diseases. The new
technologies developed in the course of the project
will be applicable in numerous other fields of
biomedical endeavour.
Each cell of an organism has a set of
chromosomes containing the heritable genetic
material that directs its development, i.e., its
genome. The genetic material of chromosomes is
DNA. Each of the paired strands of the DNA
molecule is a linear array of subunits called
nucleotides, or bases, of which there are four
types—adenine, cytosine, thymine, and guanine.
Genes are discrete stretches of nucleotides that
carry the information, which is used by the cell to
synthesize proteins.
Human genes take up only about 5 to 10% of
the DNA. Some of the remaining DNA, which
does not code for proteins, may regulate whether
or not proteins are made, but the function of most
of it is unknown.
Genome size
Haploid number
4 kb
5 kb
5 kb
6 kb
50 kb
152 kb
165 kb
267 kb
1600 kb
4600 kb
910 kb
13 Mb
97 Mb
100 Mb
180 Mb
3000 Mb
4500 Mb
400 Mb
90,000 Mb
1
1
1
1
1
1
1
1
1
1
1
16
06
05
04
23
10
22
14
This landmark of scientific achievement
represented the completion of the first stage of the
project. Initial results published by both groups in
February 2001 declared that the human genome
actually contains only about 30,000 to 40,000
genes, much fewer than originally thought. Two
types of maps were constructed—genetic linkage
maps and physical maps. Genetic linkage map
provides the relative location of genes and other
markers on the basis of how frequently genes are
inherited together; the closer genes are to each
other on a chromosome, the more likely they are
to be inherited together. Physical maps locate genes
in relation to the presence of known nucleotide
sequences that act as landmarks along the length
of a chromosome.
One such “marker” used to map the human
genome is a sequence-tagged site (STS)—a short
sequence of nucleotides that occurs only once
throughout the genome. A relatively detailed
physical map was needed before sequencing could
28 Bioethics and Biosafety
begin. Sequencing, in which the precise order of
the nucleotide sequence is determined, was the
most technically challenging part of the project.
DNA sequencing of the nematode worm
Caenorhabditis elegans and the yeast
Saccharomyces cerevisiae was completed in 1996.
The DNA sequencing of the other organisms was
completed in the following order:
(1) E. coli-1997.
(2) Fruit fly (Drosophila melanogaster) and
plant Arabidopsis thaliana—2000.
(3) The laboratory mouse (Mus musculus) and
bacterium Staphylococcus aureus—2001.
The rationale for these efforts is that many
genes with similar functions in disparate organisms
have been conserved in evolution and show
surprising similarities. Genes from simpler
organisms can thus be used to study human beings.
Another objective of the Human Genome
Project is to address the ethical, legal, and social
implications of the information obtained. Society
will derive the greatest benefit from this knowledge
only if it takes measures to prevent abuses, such
as invasion of the privacy of an individual’s genetic
background by employers, insurers,or government
agencies, or discrimination based on genetic
grounds.
The HGP was the natural culmination of the
history of genetics research. In 1911, Alfred
Sturtevant, then an undergraduate researcher in the
laboratory of Thomas Hunt Morgan, realized that
he could—and had to, in order to manage his
data—map the location of the fruit fly (Drosophila
melanogaster) genes, whose mutations Morgan
laboratory was tracking over generations.
Sturtevant’s very first gene map can be likened to
the Wright brothers’ first flight at Kitty Hawk. In
turn, the Human Genome Project can be compared
to the Apollo program bringing humanity to the
moon.
The hereditary material of all multicellular
organisms is the famous double helix of
deoxyribonucleic acid (DNA), which contains all
of our genes. DNA, in turn, is made up of four
chemical bases, pairs of which form the “rungs”
of the twisted, ladder-shaped DNA molecules. All
genes are made up of stretches of these four bases,
which are arranged in different ways and in
different lengths. HGP researchers have
deciphered the human genome in three major
ways—determining the order or “sequence”, of all
the bases in our genome’s DNA, making maps that
show the locations of genes in major sections of
all our chromosomes, and producing what are
called linkage maps, complex versions of the type
originated in early Drosophila research, through
which inherited traits (such as those for genetic
disease) can be tracked over generations.
The HGP has revealed that there are probably
somewhere between 30,000 and 40,000 human
genes, and their location can be identified now.
This ultimate product of the HGP has given the
world a resource of detailed information about the
structure, organization and function of the
complete set of human genes. This information can
be thought as the basic set of inheritable
“instructions” for the development and functioning
of a human being.
The International Human Genome Sequencing
Consortium published the first draft of the human
genome in the journal ‘Nature’ in February 2001,
with the sequence of the entire genome’s three
billion base pairs some 90 percent complete. A
startling finding of this first draft was that the
number of human genes appeared to be
significantly fewer than previous estimates, which
ranged from 50,000 genes to as many as
140,000.The full sequence was completed and
published in April 2003.
How to Sequence
The task of determining the complete sequence of
the 3,200,000,000 bases of the human genome
(30× the size of the nematode genome) was
extremely daunting at the time when the project
was formally launched. Several lines of
investigation focused on an alternative approach
Human Genome Project and its Ethical Issues 29
to characterize the human genome. For example,
complete genome sequencing may be bypassed by
selectively sequencing just expressed sequences,
obtained by extracting mRNA from a wide range
of human tissues. Large scale expressed sequence
tags (EST) projects in both the public and private
domain resulted in the collection of huge amount
of sequence information on human genes. An
international consortium to map the ESTs in the
genome, using the genetic map as a framework,
resulted in the human gene map of 35,000 genes.
This was an important and valuable milestone in
HGP. However, the sequence of most of the
mRNAs was incomplete, unknown number of
genes were missing from the collection, and there
was no information available on gene structures.
In contrast, the experience gained from the
study of smaller genomes, especially those of the
nematode and yeast illustrated the enormous
potential to obtain a complete set of genes, gene
structures and all other genetic information by
determining the complete sequence of the genome.
Furthermore, by breaking the task into manageable
segments, and using a physical map to co-ordinate
the work, it was possible to undertake projects to
sequence genomes that were far beyond the
capabilities of a simple shotgun approach. For the
human genome, therefore, the strategy adopted was
to use the landmarks provided by the genetic map,
and later the gene map, as a framework to anchor
a physical map of overlapping clones which
represented all human chromosomes. initially
much of the work was done using yeast artificial
chromosomes (YACs), a yeast cloning system,
which accepts vary large fragments and thus allows
a physical map to be built quickly over very large
distances. However, the development of new
bacterial cloning systems called BACs or PACs
(bacteria or P1 derived artificial chromosomes),
which were capable of taking large inserts (up to
250 kb) made it possible to make long range maps
directly in bacterial clones. This coupled with the
greater convenience and stability of bacterial
clones compared to YACs, resulted in the choice
of this system for construction of the physical map
to provide the tile path of clones for sequencing.
Each BACs or PACs has been sequenced
using a random shotgun approach. This approach
is essentially the same as was developed for the
early whole genome sequencing projects. DNA
from the BAC or PAC is broken up randomly into
short fragments (typically 1-2kb long), which are
sub cloned into plasmids or bacteriophage M13
cloning vector. The resulting sub clones
(transformed bacterial colonies) are picked at
random, cultured and the sub clone DNA is
extracted for use as a sequencing template. A
primer (short DNA strand) is hybridized to the
template within the vector sequence (which is
common to all clones). This provides a starting
point for DNA polymerase to synthesize new
strands of DNA by incorporating the
deoxynucleotide triphosphate (dNTPs), which are
the single base precursors of DNA.
Fluorescently labeled analogues for each base
are included in the same reaction (dideoxy NTPs
or ddNTPs); a different fluorescent tag is used for
each of the four bases. These analogues extend
the chain in a base-specific manner when they are
incorporated (and these are called “chain
terminators”). The product of the reaction is a
ladder of newly synthesized DNA fragments of
increasing size in single base increments. Each
fragment in the mixture is terminated at a specific
place, which can be identified according to its
specific fluorescent label that can be separated on
the basis of size by electrophoresis, either through
polyacrylamide “slab” gels, or more recently
through a viscous liquid matrix held in individual
capillaries (capillary gel electrophoresis). The
ladder of colored bands thus represents the
sequence of the bases in the DNA, and can be read
automatically by an automatic fluorescent detector.
The sequence of all the sub clones of a single
BAC or PAC is analyzed together. All overlap[s]
between sequences are identified, and the
individual reads are assembled onto contigs. A
consensus sequence is obtained at this stage, and
Human Genome Project and its Ethical Issues 31
Although the HGP is finished, analyses of the
data will continue for many years. An important
feature of the HGP project was the federal
government’s long-standing dedication to the
transfer of technology to the private sector. By
licensing technologies to private companies and
awarding grants for innovative research, the project
catalyzed the multibillion-dollar US biotechnology
industry, and fostered the development of new
medical applications.
Rapid progress in genome science and a
glimpse into its potential applications have spurred
observers to predict that biology will be the
foremost science of the 21st century. Technology
and resources generated by the Human Genome
Project and other genomics research are already
having a major impact on research across the life
sciences. The potential for commercial
development of genomics research presents US
industry with a wealth of opportunities, and sales
of DNA-based products and technologies in the
biotechnology industry are projected to exceed $45
billion by 2009 (Consulting Resources Corporation
Newsletter, Spring 1999).
Current and Potential Applications of
Genome Research Include the Following:
•
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Molecular medicine
Energy sources and environmental applications
Risk assessment
Bioarchaeology, anthropology, evolution, and
human migration
DNA forensics (identification)
Agriculture, livestock breeding and
bioprocessing
Molecular medicine
Improved diagnosis of disease
Earlier detection of genetic predispositions to
disease
Rational drug design
Gene therapy and control systems for drugs
Pharmacogenomics “custom drugs”
Broader applications reaching into many areas
of the economy include the following:
• Clinical medicine: Many more individualized
diagnostics and prognostics, drugs, and other
therapies.
• Agriculture and livestock: More nutritious
and healthier crops and animals.
• Industrial processes: Cleaner and more
efficient manufacturing in sectors such as
chemicals, pulp and paper, textiles, food, fuels,
metals, and minerals.
• Environmental biotechnology: Biodegradable products, new energy resources, environmental diagnostics and less hazardous cleanup
of mixed toxic-waste sites.
• DNA fingerprinting: Identification of humans
and other animals, plants and microbes;
evolutionary and human anthropological
studies; and detection of and resistance to
harmful agents that might be used in biological
warfare.
Technology and resources promoted by the
Human Genome Project are beginning to have
profound impacts on biomedical research, and
promise to revolutionize the wider spectrum of
biological research and clinical medicine.
Increasingly detailed genome maps have aided
researchers seeking genes associated with dozens
of genetic conditions, including myotonic
dystrophy, fragile X syndrome, neurofibromatosis,
diabetes types 1 and 2, inherited colon cancer,
Alzheimer’s disease and familial breast cancer.
On the horizon is a new era of molecular
medicine, characterized less by treating symptoms
and more by looking to the most fundamental
causes of disease. Rapid and more specific
diagnostic tests will make earlier treatment of
countless maladies possible. Medical researchers
will also be able to devise novel therapeutic
regimens based on new classes of drugs,
immunotherapy techniques, avoidance of
environmental conditions that may trigger disease,
and possible augmentation or even replacement
of defective genes through gene therapy.
Human Genome Project and its Ethical Issues 33
reassembling DNA fragments in their original
order. This repeated sequencing is known as
genome “depth of coverage”. Draft sequence data
is mostly in the form of 10,000 basepair-sized
fragments whose approximate chromosomal
locations are known.
In June 2000, the Human Genome Project and
Celera Genomics, a privately owned firm founded
in 1998, jointly announced the completion of the
initial sequencing of the human genome, which is
composed of about three billion nucleotide base
pairs.
Developing the Tools and Technologies for the
Success of HGP
The DOE investments described below helped to
make the Human Genome Project a success.
Substantial investments by the NIH and the
Wellcome Trust in the UK were equally important,
however, and should not be overlooked. In most
cases, the DOE achievements outlined below were
the result of basic research programs. Research is
an incremental process that learns from both the
success and failures of other research investments,
including other agencies and organizations.
Furthermore, no single instrument, technology,
reagent, or protocol made high-throughput DNA
sequencing possible, many contributors were
responsible.
DNA Sequencers
Research on capillary-based DNA sequencing
contributed to the development of two major DNA
sequencing machines—the Perkin-Elmer 3700 and
the MegaBace DNA sequencers. The MegaBace
DNA sequencer was developed initially with DOE
funds by Dr. Richard Mathies at UC, Berkeley.
The Perkin-Elmer 3700 was based, in part, on
DOE-funded research by Dr. Norman Dovichi at
the University of Alberta. These high-throughput
instruments are one of the keys to the success of
the genome project.
Fluorescent Dyes
DNA sequencing originally used radiolabeled
DNA subunits. DOE-funded research contributed
to the development of fluorescent dyes, which
increased the accuracy and safety of DNA
sequencing as well as the ability to automate the
procedures.
DNA Cloning Vectors
Before the sequencing of large DNA molecules,
they are cut into small pieces and multiplied, or
cloned into numerous copies using microbialbased “cloning” vectors. Today, the bacterial
artificial chromosome (BAC) is the most
commonly used vector for initial DNA
amplification before sequencing. These cloning
vectors were developed with DOE funds.
BAC-End Sequencing
The widely agreed-upon strategy for sequencing
the human genome is based on the use of BACs,
which carry fragments of human DNA from known
locations in the genome. DOE-funded research at
the Institute for Genomic Research in Rockville,
Maryland, and at the University of Washington
provided the sequencing community with a
complete set of over 450,000 BAC-based genetic
“markers” corresponding to a sequence tag every
3 to 4 kilobases across the entire human genome.
These markers were needed to assemble both the
draft and the final human DNA sequence.
Gene Recognition and Assembly Internet
Link (GRAIL)
Gene Recognition and Assembly Internet Link
(GRAIL) is one of the most widely used computer
programs for identifying the potential genes in
DNA sequence and for general DNA sequence
analysis. This powerful analytical tool was
developed with DOE funds by Dr. Ed Uberbacher
at Oak Ridge National Laboratory. Although a
Human Genome Project and its Ethical Issues 35
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Structural genomics: Initiatives are being
launched worldwide to generate the 3-D
structures of one or more proteins from each
protein family, thus offering clues to function
and biological targets for drug design.
Experimental methods for understanding the
function of DNA sequences and the proteins
they encode include knockout studies to
inactivate genes in living organisms, and
monitor any changes that could reveal their
functions.
Comparative genomics: Analyzing DNA
sequence patterns of humans and well-studied
model organisms side-by-side has become
one of the most powerful strategies for
identifying human genes and interpreting their
function.
2.9. FUTURE OF HGP IN THE MEDICINE
AND GENETICS
The medical industry is building upon the
knowledge, resources, and technologies emanating
from the HGP to further understanding of genetic
contributions to human health. As a result of this
expansion of genomics into human health
applications, the field of genomic medicine was
born. Genetics is playing an increasingly important
role in the diagnosis, monitoring and treatment of
diseases.
Diagnosing and Predicting Disease and
Disease Susceptibility
All diseases have a genetic component
(Fig. 2.6), whether inherited or resulting from the
body’s response to environmental stresses like
viruses or toxins. The success of the HGP has even
enabled researchers to pinpoint errors in genes—
the smallest units of heredity—that cause or
contribute to disease.
The ultimate goal is to use this information to
develop new ways to treat, cure, or even prevent
the thousands of diseases that afflict humankind.
Fig. 2.6. Diagram of human chromosome
19 showing the locations of selected defective
genes and genetic markers.
But the road from gene identification to effective
treatments is long and fraught with challenges. In
the meantime, biotechnology companies are racing
ahead with commercialization by designing
diagnostic tests to detect errant genes in people
suspected of having particular diseases or of being
at risk for developing them.
Human Genome Project and its Ethical Issues 37
analyzing and addressing the ethical, legal and
social implications of human genetics research at
the same time that the basic scientific issues are
being studied. In this way, problem areas can be
identified, and solutions developed before the
scientific information is integrated into health care
practice.
The ELSI Program is viewed as essential to
the success of the genome project in the United
States, and is supported by federal HGP funds. The
National Institutes of Health’s ‘National Human
Genome Research Institute (NHGRI)’ has
committed 5% of its annual research budget to
study ELSI issues. The US Department of Energy
Office of Energy Research, NHGRI’s partner in
the US Human Genome Project, also reserves a
portion of its funding for ELSI research and
education.
ELSI and its establishments anticipates and
addresses the implications of mapping and
sequencing of the human genomes for the
individuals and society. It also examines the ethical,
legal and social consequences of mapping and
sequencing the human genome; stimulates public
discussion of the issues; and develops policy
options, which would assure that the information
is used for the benefit of individuals and society.
The Working Group envisioned a program that
would anticipate potential problems before they
actually occur, and identify possible solutions for
the problems. It suggested a number of means for
accomplishing these goals. Specifically, it
encouraged the research community to explore and
gather data on a wide range of issues pertinent to
the human genome program that could be used to
develop educational programs, policy
recommendations or possible legislative solutions.
A number of areas for focus were identified,
including fairness in the use of genetic information,
the impact of knowledge of genetic variation on
individuals, and the privacy and confidentiality of
genetic information, to name a few.
In 1990, in response to the Working Group’s
report, the NHGRI established the ELSI Branch
(later renamed the ELSI Research Program) in its
Division of Extramural Research, and the DOE
established an ELSI program in their Office of
Energy Research. Since the beginning, these two
programs have collaborated closely, including the
joint support of the ELSI Working Group, the
development of complementary research priority
areas, and the co-funding of ELSI activities of
mutual interest.
SUMMARY
Humans are higher in the hierarchy of living
organisms because of their independent thinking
and fantasizing capacity. Thus, understanding the
human genome and its contents gives an idea about
how simple, single-celled zygote/organisms
developed into a complex individual. Human
Genome project is also called Human Genome
Initiative scientific research effort to analyze the
DNA of humans and of several lower organisms.
The project began in the United States in 1990
under the sponsorship of the US Department of
Energy and the National Institutes of Health.
Projects undertaken concurrently in Japan, the
United Kingdom, Italy, France, and Russia are
coordinated with the American effort through the
Human Genome Organization.
The project’s ultimate goal is to identify the
chromosomal location of every human gene, and
to determine each gene’s precise chemical structure
in order to elucidate its function in health and
disease. The information gathered is expected to
serve as the basic reference for research in human
biology and medicine in the 21st century, and to
provide fundamental insights into the genetic basis
of human disease. The new technologies developed
in the course of the project will be applicable in
numerous other fields of biomedical endeavour.
The total number of genes is estimated to be
30,000, which is much lower than previous
estimates of 80,000 to 140,000 that had been based
on extrapolations from gene-rich areas as opposed
to a composite of gene-rich and gene-poor areas.
38 Bioethics and Biosafety
The functions are unknown for over 50% of the
discovered genes. Less than 2% of the human
genome codes for functional proteins of the total
three billion base pairs in all cells of the body.
Repeated sequences that do not code for proteins
(“junk DNA”) make-up at least 50% of the human
genome. Repetitive sequences are thought to have
no direct functions, but they shed light on
chromosome structure and dynamics. Over time,
these repeats reshape the genome by rearranging
it, creating entirely new genes, and modifying and
reshuffling existing genes. Chromosome 1 has
most of the genes (2968) and the Y chromosome
has the fewest (231).
Genome sequencing is the term used to
describe the laboratory process of reading the order
of the four letter of the genetic alphabets (A,C,G,T)
along a strand of DNA. The various steps involved
in such efforts are as follows: Selection of suitable
sample materials for the DNA; isolation of DNA
from cells and preparation of large samples of high
quality DNA from these cells; cutting the purified
DNA at random sites into manageably sized,
overlapping pieces of the DNA sample; insertion
of these DNA pieces into packages for the
production of limitless copies of such selected
DNA; recording the order of bases for each DNA
sample piece by using DNA sequencing
techniques; determination of the overlap of each
piece, and assembling the sequences to give the
final genome of the human.
While following the above approaches, it is
necessary to make appropriate sample populations
based on the humans distribution. A primary goal
of the Human Genome project is to generate
detailed maps of the human genome. These maps
will aid in determining the location of genes within
the human genome. More specifically, they will
assign genes to their chromosomes. Two types of
maps are being generated. Genetic linkage maps
determine the relative arrangement and
approximate distances between genes and markers
on the chromosomes; physical maps specify the
physical location (in base pairs) and distance
between genes or DNA fragments with unknown
functions that are mapped to specific regions of
the chromosomes.
In the Human Genome Project, importance is
also given to the sequencing of other model
organisms. DNA sequencing of the nematode
worm Caenorhabditis elegans and the yeast
Saccharomyces cerevisiae was completed in 1996,
the bacterium Escherichia coli in 1997, the fruit
fly (Drosophila melanogaster) and the plant
Arabidopsis thaliana in 2000, and the laboratory
mouse (Mus musculus) and the bacterium
Staphylococcus aureus in 2001. The rationale
behind these findings is that many genes with
similar functions in disparate organisms have been
conserved in evolution and show surprising
similarities. Genes from simpler organisms can
thus be used to study their counterparts found in
human beings.
Another objective of the Human Genome
Project is to address the ethical, legal, and social
implications of the information obtained. Society
will derive benefit from this knowledge only if it
takes measures to prevent abuses, such as invasion
of the privacy of an individual’s genetic
background by employers, insurers, or government
agencies, or discrimination based on genetic
grounds.
Large number of advancement made in the
diverse fields including molecular biology, genetic
engineering and sequencing provided a great
impetus to the Human Genome Project. These
technological developments dramatically
decreased the cost of DNA sequencing, while
increasing its speed and efficiency. For example,
it took four years for the international Human
Genome Project to produce the first billion base
pairs of sequence, and less than four months to
produce the second billion base pairs. In the month
of January, 2003, the DOE team sequenced 1.5
billion bases. The cost of sequencing has dropped
dramatically since the project began and is still
dropping rapidly. Other major factors involved in
cost and time reduction were greatly improved