HUMAN GENOME

PROJECT
 At a glance
• The Human Genome
– Salient Features of Human Genome
• What was Human Genome Project(HGP)
– Milestones
– Goals of Human Genome Project
– Issues of concern
– Future Challenges
• Vectors for Large-Scale Genome Project
– Yeasts artificial chromosomes
– Bacterial artificial chromosome
– Difference between YAC and BAC
 The Human Genome
• The human genome is the complete set of genetic
information for humans (Homo sapiens).
• The human genome is by far the most complex and largest
genome.
• Its size spans a length of about 6 feet of DNA, containing
more than 30,000 genes.
• The DNA material is organized into a haploid chromosomal
set of 22 (autosome) and one sex chromosome (X or Y).

Male Female
 Human Genome Sequencing 2/11/2001
22 autosome + 2 sex chromosomes

From NCBI
Salient Features of Human Genome:
 Human genome consists the information of 24
chromosomes (22 autosome + X chromosome + one Y
chromosome); in Homo sapiens 2n = 2x = 46
 The human genome contains over 3 billion nucleotide pairs.
 Human genome is estimated to have about 30,000 genes .
 Average gene consists of 3000 bases. But sizes of genes
vary greatly, with the largest known human gene encoding
dystrophin containing 2.5 million base pairs.
 Only about 3 %of the genome encodes amino acid
sequences of polypeptides and rest of it junk (repetitive
DNA).
 The functions are unknown for over 50% of the discovered
genes.
Continue………
The repetitive sequences makeup very large portion of
human genome. Repetitive sequences have no direct
coding function but they shed light on the chromosome
structure, dynamics and evolution.
Chromosome 1 has most genes (2968) and Y chromosome
has the lowest (231).
Almost all nucleotide bases are exactly the same in all
people. Genome sequences of different individuals differ
for less than 0.2% of base pairs.
Most of these differences occur in the form of single
base differences in the sequence. These single base
differences are called single nucleotide polymorphisms
(SNPs). One SNP occurs at every ~ 1,000 bp of human
genome. About 85% of all differences in human DNAs are
due to SNPs.
Human Chromosome 1 Genetic Map
 What was Human Genome Project(HGP)
• The Human Genome Project
was an international research
effort to determine the
sequence of the human
genome and identify the
genes that it contains.

• The US Human Genome

Project is a 13 year effort,
which is coordinated by the
– Department of Energy (DOE) and
– National Institutes of Health
(NIH).
 Milestones
1986 The birth of the Human Genome Project.
1990 Project initiated as joint effort of US Department of
Energy and the National Institute of Health.
1994 Genetic Privacy Act: to regulate collection, analysis,
storage and use of DNA samples and genetic information
is proposed.
1996 Welcome Trust joins the project.
1998 Celera Genomics (a private company founded by Craig Venter)
formed to sequence much of the human genome in 3 years.
1999 Completion of the sequence of Chromosome 22-the first
human chromosome to be sequenced.
2000 Completion of the working draft of the entire human
genome.
2001 Analysis of the working draft are published.
2003 HGP sequencing is completed and Project is declared finished
two years ahead of schedule.
 Goals of Human Genome Project
1. To identify all the genes in human DNA.
2. To develop a genetic linkage map of human genome.
3. To obtain a physical map of human genome.
4. To develop technology for the management of human
genome information.
5. To know the function of genes.
6. Determine the sequences of the 3 billion chemical
base pairs that make up human DNA.
7. Store this information in public databases.
8. Develop tools for data analysis.
9. Transfer related technologies to the private sectors.
 ISSUES OF CONCERN
Ethical, Legal and Social issues of the Human Genome
Project
• Fairness in the use of genetic information.
• Privacy and confidentiality of genetic information.
• Psychological impact, stigmatization, and discrimination.
• Reproductive issues.
• Clinical issues.
• Uncertainties associated with gene tests for susceptibilities and
complex conditions.
• Fairness in access to advanced genomic technologies.
• Conceptual and philosophical implications.
• Health and environmental issues.
• Commercialization of products.
• Education, Standards, and Quality control.
• Patent issues.
Future Challenges: What We Still Don’t Know

1. Gene number, exact locations, and functions

2. Gene regulation
3. Chromosomal structure and organization
4. Non-coding DNA types, amount, distribution, information
content, and functions
5. Coordination of gene expression, protein synthesis, Proteomes
and post-translational events
6. Predicted vs experimentally determined gene function
7. Evolutionary conservation among organisms
8. Disease-susceptibility prediction based on gene sequence
variation
9. Genes involved in complex traits and multigene diseases
10. Developmental genetics, genomics
 Vectors for Large-Scale Genome Project
A vector is a DNA molecule that has the ability to replicate
in an appropriate host cell, and into which the DNA insert is
integrated for cloning.
A vector must have a origin of DNA replication (ori).
The vector is a vehicle or carrier which is used for cloning
foreign DNA in bacteria.
For genome sequencing, first DNA fragments of the
genome must be cloned in appropriate vectors. Two of the
most popular vector:
1. Yeast artificial chromosomes (YACs) and
2. Bacterial artificial chromosomes (BACs)
 Yeasts artificial chromosomes (YACs):
Yeast artificial chromosomes (YACs) are genetically engineered
chromosomes derived from the DNA of the yeast, Saccharomyces
cerevisiae, which is then ligated into a bacterial plasmid.
• YACs were very useful in mapping the human genome because they
could accommodate hundreds of thousands of kilo bases each.
• YACs containing a mega base or more are known as "mega YACs."

A YAC can be considered as self replicating element, because it includes

three specific DNA sequences:
1. TEL: The telomere which is located at each chromosome end,
protects the chromosome's ends from degradation by nucleases.
2. CEN: The centromere which is the attachment site for mitotic spindle
fibers and necessary for segregation of sister chromotids to opposite
poles of the dividing yeast cell. The centromere is placed in adjacent
to the left telomere, and a huge piece of human (or any other) DNA
can be placed in between the centromere and the right telomere.
Continue………
3. ORI: Replication origin sequences which are specific DNA sequences
that allow the DNA replication.

It also contains few other specific sequences like:

Selectable markers (A and B) that allow the easy isolation of yeast
cells that have taken up the artificial chromosome.
Recognition site for the two restriction enzymes EcoRI and BamHI.
Cloning genomic DNA into a YAC
1. Genomic DNA is partially
digested with a restriction
enzyme (EcoRI).
2. The YAC is digested by the
two restriction enzymes EcoRI
and BamHI.
3. Those two elements
recombine at the EcoRI sites of
YAC and are covalently linked
by the DNA ligase.
4. A recombinant YAC vector, a
yeast artificial chromosome
with genomic DNA inserted, is
produced. Then YACs vector
can be introduced into yeast
cells and generated an
unlimited number of copies. Fig: Cloning of genomic DNA into a YAC
 Bacterial artificial chromosome (BAC)
A bacterial artificial chromosome (BAC) is an engineered
DNA molecule, used to clone DNA segment in bacterial
cells (E. coli).
It is based on a well-known natural F plasmid (inhabits E.
coli cells). This plasmid allows conjugation between
bacterial cells.
• Segments of an organism's DNA, ranging from 150 to
about 300 kilo base pairs, can be inserted into BACs.
• These vectors are able to maintain in stable state in
vivo and in vitro.
• Their copy number is about two per cell.
• Extensively used in analysis of large genomes but the
main disadvantage of BAC vectors is some what
laborious construction of BAC libraries.
Common gene components
Bacterial artificial chromosome is another cloning vector system in E.coli
(pBAC108L), developed by Melsimon and his colleagues in 1992, have
 HindIII and BamHI: the
cloning sites
 CmR: the chloramphenicol
resistance gene, used as a
selection tool.
 oriS: the origin of
replication
 repE: for plasmid
replication and regulation of
copy number.
 ParA and ParB: the genes
governing partition of plasmids
to daughter cells during
division and ensures stable
maintenance of the BAC. Fig: Map of the BAC vector, pBAC108L
In some conjugation events, the F-plasmid itself is
transferred from a donor F+ cell to a recipient F- cell,
converting the letter to an F+ cell.

In other events, a small piece of host DNA is transferred as

an insert in the F (which is called an F' plasmid if it has an
insert of foreign DNA). And in still other events, the F'
plasmid inserts into the host chromosome and mobilizes the
whole chromosome to pass from the donor cell to the
recipient cell. Thus, because the E. coli chromosome
contains over 4 million bp, the F plasmid can obviously
accommodate a large insert of DNA.
Cloning genomic DNA into a BAC
1. Genomic DNA is isolated
from a desired source and
used restriction enzymes
to cleave the target DNA
into fragments.
2. The BAC is digested by
restriction enzymes in the
cloning sites HindIII and
BamHI.
3. Those two elements
recombine by the DNA
ligase and attach into a
host bacterium.
4. As the bacterial cells
grow and divide, they
amplify the BAC DNA,
which can then be isolated
and used in sequencing
DNA. Fig: BAC as a Cloning vector
 Difference between YAC and BAC
as vector of genome sequencing
Yeast artificial chromosomes (YACs) Bacterial artificial chromosome (BAC)
1. Yeast artificial chromosomes (YACs) are 1. A bacterial artificial chromosome (BAC)
genetically engineered chromosomes is an engineered DNA molecule, used to
derived from the DNA of the clone DNA segment in bacterial cells (E.
yeast, Saccharomyces cerevisiae. coli).
2. YAC’s are used for cloning very large 2. These vectors are used to clone the DNA
(1000-2000kb) DNA segments. inserts up to 300kb.
3. They are inefficient. 3. They are inefficient.
4. Unlike BAC library, it is not so hard to 4. It is very hard to construct BAC library.
construct YAC library.
5. They are unstable. 5. They are more stable.
6. They tend to contain scrambled inserts, 6. They contain pure inserts.
i.e. composites of DNA fragments from
more than one site.
7. The linear YACs, which tend to break 7. The circular, super coiled BACs resist
under shearing forces. breakage.
8. They are hard to isolate from yeast cells. 8. They are easy to isolate.
 References
• Weaver RF 2005. Molecular Biology. McGraw-Hill
International edition, NY.
• Gardner EJ, MJ Simmons and DP Snustad 1991.
Principles of Genetics. John Wiley and Sons Inc,
NY.
• Gupta, P.K. 2007. Genetics. Rastogi Publications,
Meerut.
• Singh, B.D. 2007. Fundamentals of Genetics.
Kalyani Publishers, Ludhiana.
• Internet