Human Genome Project

Begun in 1990, the U.S. Human Genome Project is a 13-year effort coordinated by the
U.S. Department of Energy and the National Institutes of Health. The project originally
was planned to last 15 years, but effective resource and technological advances have
accelerated the expected completion date to 2003. Project goals are to

■ identify all the approximate 30,000 genes in human DNA,

■ determine the sequences of the 3 billion chemical base pairs that make up human
DNA,
■ store this information in databases,
■ improve tools for data analysis,
■ transfer related technologies to the private sector, and
■ address the ethical, legal, and social issues (ELSI) that may arise from the project.

Recent Milestones:
■ June 2000 completion of a working draft of the entire human genome
■ February 2001 analyses of the working draft are published
Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001
Genoteca genomica o di cDNA

CGAP
Cell-fusion techniques applied to
human and mouse cells produce
colonies, each of which contains a
full mouse genome plus a few human
chromosomes (blue). A fibroblast is a
cell of fibrous connective tissue.
Making radiation hybrids
using X rays. Fragments
of human chromosomes
integrate into rodent
chromosomes. A panel of
different radiation
hybrids is analyzed for
cotransfer of human
markers, which can
indicate linkage
Using sequence-
tagged sites (STSs) to
order overlapping
clones (YACs, in this
example) into a
contig. Five different
YACs are tested to
determine which
STSs they contain
(top), and these data
are used to assemble
a physical map
(bottom).
1. Clonaggio in PAC o BAC (circa 150-200 Kb)
2. Ordinamento per cromosoma
3. Subclonaggio in plasmidi di ciascun PAC/BAC (meno di 1 Kb)
4. Sequenziamento
5. Assemblaggio delle sequenze
October 2002 marks the 20th
anniversary of the creation of
GenBank. GenBank has grown
from 680,338 base pairs in 1982
to 22 billion base pairs in 2002. In
1984, GenBank was distributed on
magnetic tape to 120 institutions
and had a daily average of 5
online users. Today, over 30,000
people per day access GenBank
online.
Genoteca genomica o di cDNA

CGAP
The synthesis of double-stranded
cDNA from mRNA.
A short oligo(dT) chain is hybridized to
the poly(A) tail of an mRNA strand.
The oligo(dT) segment serves as a
primer for the action of reverse
transcriptase, which uses the mRNA as
a template for the synthesis of a
complementary DNA strand. The
resulting cDNA ends in a hairpin loop.
When the mRNA strand has been
degraded by treatment with NaOH, the
hairpin loop becomes a primer for DNA
polymerase I, which completes the
paired DNA strand. The loop is then
cleaved by S1 nuclease (which acts
only on the single-stranded loop) to
produce a double-stranded cDNA
molecule
 EST: expressed sequence tags

5’ mRNA

cDNA

cDNA

ESTs

5’ mRNA
Cells and Gene Expression I
Although every cell contains the full set of 100,000 genes, only about one tenth of them are expressed in
any particular type of cell. Cells look and act the way they do because of the specific genes that they
express and the amounts of gene products produced. For example, a muscle cell, a skin cell, and a nerve
cell, could be distinguished by their gene expression profiles
 Human Genome Project

Begun in 1990, the U.S. Human Genome Project is a 13-year effort coordinated by the
U.S. Department of Energy and the National Institutes of Health. The project originally
was planned to last 15 years, but effective resource and technological advances have
accelerated the expected completion date to 2003. Project goals are to

■ identify all the approximate 30,000 genes in human DNA,

■ determine the sequences of the 3 billion chemical base pairs that make up human
DNA,
■ store this information in databases,
■ improve tools for data analysis,
■ transfer related technologies to the private sector, and
■ address the ethical, legal, and social issues (ELSI) that may arise from the project.

Recent Milestones:
■ June 2000 completion of a working draft of the entire human genome
■ February 2001 analyses of the working draft are published
Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001
 What does the draft human
genome sequence tell us?

By the Numbers
• The human genome contains 3164.7 million chemical nucleotide bases (A, C, T,
and G).
• The average gene consists of 3000 bases, but sizes vary greatly, with the
largest known human gene being dystrophin at 2.4 million bases.
• The total number of genes is estimated at 30,000 to 35,000 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.
• Almost all (99.9%) nucleotide bases are exactly the same in all people.
• The functions are unknown for over 50% of discovered genes.

Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001
 What does the draft human
genome sequence tell us?
Variations and Mutations
• Scientists have identified about 1.4 million locations where single-base DNA
differences (SNPs) occur in humans. This information promises to revolutionize the
processes of finding chromosomal locations for disease-associated sequences and
tracing human history.

• The ratio of germline (sperm or egg cell) mutations is 2:1 in males vs females.
Researchers point to several reasons for the higher mutation rate in the male germline,
including the greater number of cell divisions required for sperm formation than for eggs.

Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001
 Future Challenges:
What We Still Don’t Know
• Gene number, exact locations, and functions
• Gene regulation
• DNA sequence organization
• Chromosomal structure and organization
• Noncoding DNA types, amount, distribution, information content, and functions
• Coordination of gene expression, protein synthesis, and post-translational events
• Interaction of proteins in complex molecular machines
• Predicted vs experimentally determined gene function
• Evolutionary conservation among organisms
• Protein conservation (structure and function)
• Proteomes (total protein content and function) in organisms
• Correlation of SNPs (single-base DNA variations among individuals) with health and
disease
• Disease-susceptibility prediction based on gene sequence variation
• Genes involved in complex traits and multigene diseases
• Complex systems biology including microbial consortia useful for environmental
restoration
• Developmental genetics, genomics
Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001
 Next Step in Genomics
• Transcriptomics involves large‑scale analysis of messenger RNAs (molecules that
are transcribed from active genes) to follow when, where, and under what conditions
genes are expressed.

• Proteomics—the study of protein expression and function—can bring researchers

closer than gene expression studies to what’s actually happening in the cell.

• 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.

• Knockout studies are one experimental method for understanding the function of
DNA sequences and the proteins they encode. Researchers inactivate genes in living
organisms and monitor any changes that could reveal the function of specific genes.

• 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.
Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001
 Clonaggio di geni-malattia

Base biochimica nota Base biochimica ignota

= clonaggio posizionale

Oligonucleotidi
Anticorpi Anomalie cromosomiche Analisi di linkage
Complementazione

Definizione della regione

del genoma in cui mappa
il gene responsabile

Identificazione del gene

Positional cloning
Positional cloning