THE HUMAN GENOM PROJECT

Outline
Introduction/Definition
History
Application
Future direction
Conclusion

Introduction
The Human Genome Project (HGP) is an international, interdisciplinary, scientific
research project aimed at determining the sequence of chemical base pairs which
make up human DNA, mapping the entire human genome, and identifying its
complex structures and functions. The outcomes of the project have been established
as open and accessible to all.
Sequencing the human genome in its entirety substantially furthers our
understanding of human evolution, the causes and mechanisms of disease, and the
complex interactions between genes and environment. Challenges over the next
decade include interpreting the contents of all the sequenced genes and
understanding how they function. Genome-based research will eventually enable
medical science to develop highly effective diagnostic tools, to better understand
health needs based on individual genetic make-ups, and to design new, personalized
treatments for disease.

History
The Human Genome Project (HGP) was first publically advocated in 1984 by
Renato Dulbecco. In 1985, a dozen experts convened in Santa Cruz, California, to
discuss the feasibility and professional implications of the HGP and concluded that
it would be possible, but challenging. The HGP was controversial among these
experts. Those opposing the HGP argued that sequencing the human genome was
not worthwhile because it is mostly junk, which current technology was not up to
the task, and that manually sequencing the genome was too mundane of a job to be
attractive to talented people. The HGP continued to be objected to throughout the
1980s by the majority of biologists, as well as the National Institutes of Health.
Crucial support for the HGP came from the US Department of Energy in 1986, which
supported the project because they sought data on protecting the genome from gene-
mutating effects of radiation. Consequently, an early genome project was established
in 1987 (National Human Genome Research Institute – NHGRI 2012) under the
direction of the National Institutes of Health and the US Department of Energy,
entailing a 15-year, $3 billion, plan to complete the human genome sequence.
Parallel to the US government sponsored HGP, the American researcher Craig
Venter, through his firm Celera Genomics, announced in 1998 his intention to build
a unique genome-sequencing facility to determine the sequence of the human
genome over a 3-year period. The first analyses of the draft human genome sequence
were reported in the February 2001 issues of Science and Nature. The Nature
publications included initial sequence analyses generated by the publicly sponsored
HGP (see Lander et al. 2001), while the Science publications focused on the draft
sequence reported by the private company Celera Genomics (see Venter et al. 2001).
In 2003, the HGP was declared complete (Human Genome Project Information
Archive – HGPIA 2015).
The studies announcing completion reported 99% of the euchromatic human genome
with 99.99% accuracy (International Human Genome Sequencing Consortium –
IHGSC 2004), followed by a major quality assessment of the human genome
sequence that indicated over 92% of sampling exceeded 99.99% accuracy (Schmutz
et al. 2004). At the time, the human genome was estimated to contain approximately
20,000–25,000 genes. The main differences between the draft (Lander et al. 2001;
Venter et al. 2001) and finished versions of the human genome sequence (IHGSC
2004) were the percentages of genome covered, the number of gaps, and the error
rates. The draft sequence covered 90% of the genome at an error rate of 1 in 1,000
base pairs, and there were over 150,000 gaps, with only 28% of the genome reaching
finished standard. In the finished version, there were less than 400 gaps, and 99% of
the genome was sequenced with an error rate of less than 1 in 10,000 base pairs.
These differences are significant for scientists using the sequence to conduct
research (NHGRI 2010).
After the human genome sequencing was complete, the US Department of Justice
filed a court brief stating that genes should not be eligible for patents because they
are products of nature. Thus, the human genome database is publicly available to
anyone (see The Genome Database – GDB, gdb. org). Analyses of the HGP data are
ongoing (see HGPIA 2015).
Applications
Sequencing the human genome in its entirety substantially furthers our
understanding of human evolution, the causes and mechanisms of disease, and the
complex interactions between genes and environment. The HGP importantly
contributes to health improvement in many ways. For example, individualized
analysis based on a person’s unique DNA has the potential to be a powerful tool for
medical prevention and treatment (NHGRI 2010). Physicians will be able to better
predict future risks of illness onset and potentially harmful behaviors that impact
each individual. Nurses, genetic counselors, and other healthcare professionals will
be able to focus their efforts on aspects that are most likely to maintain or improve
individual patients’ health. These aspects may include personalized dietary and
lifestyle changes and targeted medical monitoring.
Understanding of prevalent diseases such as diabetes, heart disease, and
schizophrenia at the genetic and molecular level may revolutionize healthcare
through earlier, more precise, detection and, therefore, intervention. Application of
new and more effective drugs based on the completed genome sequencing are at
least 10–15 years in the future, although more than 350 biotech products – many
based on the HGP – are currently in clinical trials (NHGRI 2010). Usually, it takes
over a decade for companies to conduct the clinical studies that are needed for
marketing approval from reputable institutions. Testing for health risks and genetic
predispositions to disease, however, will arrive more quickly – in particular abilities
to predict individual future health risks and implement an enhanced approach to
preventive medicine. Moreover, researchers and physicians will be able to better
determine which drugs will provide the best outcomes for particular individuals,
based on their genetic make-up (NHGRI 2010).
One of the largest challenges over the next decade will be interpreting the contents
of all the sequenced genes and understanding how they function – including their
role in human health and pathology (NHGRI 2010). Genome-based research will
eventually enable medical science to develop highly effective diagnostic tools, to
better understand health needs based on individual genetic make-ups, and to design
new, personalized treatments for disease.

Future Directions
The HGP and consequent deeper knowledge of our genome has revolutionized the
practice of medicine, inspiring several large-scale data acquisition initiatives such as
the 1000 Genomes Project, the Chimpanzee Genome Project, the Neanderthal
Genome Project, and the Cancer Genome Atlas. Starting in 2008, an international
research initiative called the 1000 Genomes Project set out to create a complete and
detailed catalogue of human genetic variations (www. 1000genomes.org). Such a
catalogue will be useful in association studies that link genetic variation to disease.
Another related project is the Chimpanzee Genome Project, which was created in
2003 and aimed to determine the DNA sequence of the chimpanzee genome.
Comparing the genomes of humans and other apes will shed new light on what
makes humans genetically distinct from, and similar to, other species.
Similarly, the Neanderthal Genome Project is an endeavor to sequence the
Neanderthal genome (www.neander tal.ensemblgenomes.org). The Neanderthal
Genome Project published their results in 2010 in Science, detailing an initial draft
of the Neanderthal genome based on the analysis of four billion base pairs of
Neanderthal DNA.
Other projects inspired by the HGP include the Human Brain Project and the
emerging Human Proteome Project, both of which were recently launched and hold
great promise for the future of medicine and psychology (reviewed in Hood and
Rowen 2013).
 CLONING

Outline
Definition
Purpose
basic steps of DNA cloning
Uses of DNA cloning
Gene libraries

Introduction
DNA cloning is a molecular biology technique that makes many identical copies of
a piece of DNA, such as a gene.
In a typical cloning experiment, a target gene is inserted into a circular piece of DNA
called a plasmid.
The plasmid is introduced into bacteria via a process called transformation, and
bacteria carrying the plasmid are selected using antibiotics.
Bacteria with the correct plasmid are used to make more plasmid DNA or, in some
cases, induced to express the gene and make protein.
When you hear the word “cloning,” you may think of the cloning of whole
organisms, such as Dolly the sheep. However, all it means to clone something is to
make a genetically exact copy of it. In a molecular biology lab, what’s most often
cloned is a gene or other small piece of DNA.
If your friend the molecular biologist says that her “cloning” isn’t working, she's
almost certainly talking about copying bits of DNA, not making the next Dolly!

Overview of DNA cloning

DNA cloning is the process of making multiple, identical copies of a particular piece
of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of
interest (perhaps a gene for a medically important human protein) is first inserted
into a circular piece of DNA called a plasmid. (A plasmid is an example of a vector,
a mechanism for storing and transporting cloned DNA segments. Plasmids are small
circles of DNA in bacterial cells that are naturally present in addition to the bacteria’s
other DNA. They contain only a few of the thousands of genes that are present in a
bacterium genome and can be copied many times in a single bacterial cell. In library
construction some of the enzyme cut DNA pieces will combine with the cut plasmids
and form recombinant DNA ). The insertion is done using enzymes that “cut and
paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled
out of fragments from multiple sources.

Diagram showing the construction of a recombinant DNA molecule.

A circular piece of plasmid DNA has overhangs on its ends that match those of a
gene fragment. The plasmid and gene fragment are joined together to produce a
gene-containing plasmid. This gene-containing plasmid is an example of
recombinant DNA, or a DNA molecule assembled from DNA from multiple sources.

Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the
plasmid are selected and grown up. As they reproduce, they replicate the plasmid
and pass it on to their offspring, making copies of the DNA it contains.
What is the point of making many copies of a DNA sequence in a plasmid? In some
cases, we need lots of DNA copies to conduct experiments or build new plasmids.
In other cases, the piece of DNA encodes a useful protein, and the bacteria are used
as “factories” to make the protein. For instance, the human insulin gene is expressed
in E. coli bacteria to make insulin used by diabetics.

Steps of DNA cloning

DNA cloning is used for many purposes. For example, DNA cloning can be used to
synthesize a protein (such as human insulin) in bacteria. The basic steps are:
Cut open the plasmid and "paste" in the gene. This process relies on restriction
enzymes (which cut DNA) and DNA ligase (which joins DNA).
Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that
took up the plasmid.
Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the
protein. Harvest the protein from the bacteria and purify it.
Let's take a closer look at each step.

1. Cutting and pasting DNA

How can pieces of DNA from different sources be joined together? A common
method uses two types of enzymes: restriction enzymes and DNA ligase.
A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target
sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes
produce cut ends with short, single-stranded overhangs. If two molecules have
matching overhangs, they can base-pair and stick together. However, they won't
combine to form an unbroken DNA molecule until they are joined by DNA ligase,
which seals gaps in the DNA backbone.

Our goal in cloning is to insert a target gene (e.g., for human insulin) into a plasmid.
Using a carefully chosen restriction enzyme, we digest:
The plasmid, which has a single cut site
The target gene fragment, which has a cut site near each end
Then, we combine the fragments with DNA ligase, which links them to make a
recombinant plasmid containing the gene.
Diagram depicting restriction digestion and ligation in a simplified schematic.

We start with a circular bacterial plasmid and a target gene. On the two ends of the
target gene are restriction sites, or DNA sequences recognized by a particular
restriction enzyme. In the plasmid, there is also a restriction site recognized by that
same enzyme, right after a promoter that will drive expression in bacteria.

Both the plasmid and the target gene are (separately) digested with the restriction
enzyme. The fragments are purified and combined. They have matching "sticky
ends," or single-stranded DNA overhangs, so they can stick together.

The enzyme DNA ligase joins the fragments with matching ends together to form a
single, unbroken molecule of DNA. This produces a recombinant plasmid that
contains the target gene.

2. Bacterial transformation and selection

Plasmids and other DNA can be introduced into bacteria, such as the harmless E.
coli used in labs, in a process called transformation. During transformation, specially
prepared bacterial cells are given a shock (such as high temperature) that encourages
them to take up foreign DNA.
The DNA produced by ligation (which may be a mix of desired plasmids, side-
product plasmids, and linear DNA pieces) is added to bacteria. The bacteria are given
a heat shock, which makes them more apt to take up DNA by transformation.
However, only a tiny minority of the bacteria will successfully take up a plasmid.

A plasmid typically contains an antibiotic resistance gene, which allows bacteria to

survive in the presence of a specific antibiotic. Thus, bacteria that took up the
plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without
a plasmid will die, while bacteria carrying a plasmid can live and reproduce. Each
surviving bacterium will give rise to a small, dot-like group, or colony, of identical
bacteria that all carry the same plasmid.
Some critics of genetic engineering claim that the potential risk of providing an
opportunity for organisms in nature to gain antibiotic resistance by taking up the
plasmid outweighs the potential benefits of this technology.
Left panel: Diagram of plasmid, showing that it contains an antibiotic resistance
gene.

Right panel: all the bacteria from the transformation are placed on an antibiotic plate.
Bacteria without a plasmid will die due to the antibiotic. Each bacterium with a
plasmid makes a colony, or a group of clonal bacteria that all contain the same
plasmid. A typical colony looks like a small, whitish dot the size of a pinhead.
Not all colonies will necessarily contain the right plasmid. That’s because, during a
ligation, DNA fragments don’t always get “pasted” in exactly the way we intend.
Instead, we must collect DNA from several colonies and see whether each one
contain the right plasmid. Methods like restriction enzyme digestion and PCR are
commonly used to check the plasmids.

3. Protein production
Once we have found a bacterial colony with the right plasmid, we can grow a large
culture of plasmid-bearing bacteria. Then, we give the bacteria a chemical signal that
instructs them to make the target protein.
The bacteria serve as miniature “factories," churning out large amounts of protein.
For instance, if our plasmid contained the human insulin gene, the bacteria would
start transcribing the gene and translating the mRNA to produce many molecules of
human insulin protein. More about expressing human genes in bacteria
A selected colony is grown up in a large culture (e.g., a 1-liter flask). The bacteria
in the large culture are induced to express the gene contained in the plasmid, causing
the gene to be transcribed into mRNA, and the mRNA to be translated into protein.
The protein encoded by the gene accumulates inside of the bacteria.
Once the protein has been produced, the bacterial cells can be split open to release
it. There are many other proteins and macromolecules floating around in bacteria
besides the target protein (e.g., insulin). Because of this, the target protein must be
purified, or separated from the other contents of the cells by biochemical techniques.
The purified protein can be used for experiments or, in the case of insulin,
administered to patients.
The recombinant insulin made by pharmaceutical companies is in fact generated in
bacteria, and it's expressed from a plasmid that bears a modified human insulin gene.
The insulin made by the bacteria is purified using a form of column purification.
However, producing biologically active insulin requires some additional steps.
Insulin is made as a single polypeptide chain. However, disulfide bonds must form
within the chain, and part of it must be snipped out to form two separate chains (held
together only by the disulfide bonds). Only then is the insulin biologically active,
that is, able to act as a hormone in a patient's body.
Production of insulin that can be used by diabetics requires that these additional steps
be incorporated into the protein purification procedure, so that the insulin protein
takes on its correct three-dimensional form.

NB:-Humans and bacteria share the same genetic code, meaning that a human gene
can be transcribed and translated in bacteria.
However, in order for a human gene to be expressed in bacteria, it must have one
important modification. Specifically, it must lack introns. Introns are intervening
sequences found in many human genes, and they are removed from eukaryotic RNA
transcripts by splicing to make a mature mRNA. Bacteria do not have introns and
cannot splice RNA transcripts.
How can we get a version of a human gene with the introns removed? An intron-less
version of a human gene can be made from the mRNA that encodes the gene, using
the enzyme reverse transcriptase. Reverse transcriptase synthesizes a DNA strand
using an mRNA strand as a template. After an additional step to the DNA double-
stranded, an intron-less version of the gene (called a cDNA) has been produced.

Restriction enzymes & DNA ligase

 Restriction enzymes are DNA-cutting enzymes.

 Each enzyme recognizes one or a few target sequences and cuts DNA at or
near those sequences.
 Many restriction enzymes make staggered cuts, producing ends with single-
stranded DNA overhangs. However, some produce blunt ends.
 DNA ligase is a DNA-joining enzyme. If two pieces of DNA have matching
ends, ligase can link them to form a single, unbroken molecule of DNA.
 In DNA cloning, restriction enzymes and DNA ligase are used to insert genes
and other pieces of DNA into plasmids.

Restriction enzymes
Restriction enzymes are found in bacteria (and other prokaryotes). They recognize
and bind to specific sequences of DNA, called restriction sites. Each restriction
enzyme recognizes just one or a few restriction sites. When it finds its target
sequence, a restriction enzyme will make a double-stranded cut in the DNA
molecule. Typically, the cut is at or near the restriction site and occurs in a tidy,
predictable pattern.

Q:Why do bacteria have restriction enzymes?

It’s thought that restriction enzymes evolved as a defense mechanism, allowing
bacteria to chop up potentially harmful foreign DNA (e.g., DNA from bacteria-
infecting viruses).
As an example of how a restriction enzyme recognizes and cuts at a DNA sequence,
let's consider EcoRI, a common restriction enzyme used in labs. EcoRI cuts at the
following site:

When EcoRI recognizes and cuts this site, it always does so in a very specific pattern
that produces ends with single-stranded DNA “overhangs”:

If another piece of DNA has matching overhangs (for instance, because it has also
been cut by EcoRI), the overhangs can stick together by complementary base
pairing. For this reason, enzymes that leave single-stranded overhangs are said to
produce sticky ends. Sticky ends are helpful in cloning because they hold two pieces
of DNA together so they can be linked by DNA ligase.
NB: Not all restriction enzymes produce sticky ends. Some are “blunt cutters,”
which cut straight down the middle of a target sequence and leave no overhang. The
restriction enzyme SmaI is an example of a blunt cutter:

Blunt-ended fragments can be joined to each other by DNA ligase. However, blunt-
ended fragments are harder to ligate together (the ligation reaction is less efficient
and more likely to fail) because there are no single-stranded overhangs to hold the
DNA molecules in position.
NB:- Restriction enzymes are named from the prokaryotic species they are isolated
from. For example, enzymes isolated from Escherichia coli bacteria would begin
with Eco (as in EcoRI).

DNA ligase
DNA ligase is an enzyme used in DNA replication. In DNA replication, ligase’s job
is to join together fragments of newly synthesized DNA to form a seamless strand.
The ligases used in DNA cloning do basically the same thing. If two pieces of DNA
have matching ends, DNA ligase can join them together to make an unbroken
molecule.

How does DNA ligase do this? Using ATP as an energy source, ligase catalyzes a
reaction in which the phosphate group sticking off the 5’ end of one DNA strand is
linked to the hydroxyl group sticking off the 3’ end of the other. This reaction
produces an intact sugar-phosphate backbone.

Uses of DNA cloning

DNA molecules built through cloning techniques are used for many purposes in
molecular biology. A short list of examples includes:
Biopharmaceuticals.
DNA cloning can be used to make human proteins with biomedical applications,
such as the insulin mentioned above. Other examples of recombinant proteins
include human growth hormone, which is given to patients who are unable to
synthesize the hormone, and tissue plasminogen activator (tPA), which is used to
treat strokes and prevent blood clots. Recombinant proteins like these are often made
in bacteria.
Gene therapy.
In some genetic disorders, patients lack the functional form of a particular gene.
Gene therapy attempts to provide a normal copy of the gene to the cells of a patient’s
body. For example, DNA cloning was used to build plasmids containing a normal
version of the gene that's nonfunctional in cystic fibrosis. When the plasmids were
delivered to the lungs of cystic fibrosis patients, lung function deteriorated less
quickly.
Gene analysis.
In basic research labs, biologists often use DNA cloning to build artificial,
recombinant versions of genes that help them understand how normal genes in an
organism function. See an example

Gene Libraries
The step following DNA extraction of an organism is the construction of a library to
organize that DNA. A gene library can be defined as a collection of living bacteria
colonies that have been transformed with different pieces of DNA from the organism
that is the source of the gene of interest. If a library is to have a colony of bacteria
for every gene, it will consist of tens of thousands of colonies or clones.

Library Construction
Constructing a gene library requires not only the extracted DNA, but also restriction
enzymes and a plasmid. A gene library is made by cutting the extracted DNA into
gene size pieces using restriction enzymes. These enzymes read the nucleotide
sequence of the DNA, recognize specific sequences and then cut the DNA sequence
by breaking the bonds between nucleotides in a DNA strand. The small DNA pieces
are mixed into a test tube containing bacteria plasmids that have been cut with the
same restriction enzyme.

A plasmid is an example of a vector, a mechanism for storing and transporting

cloned DNA segments. Plasmids are small circles of DNA in bacterial cells that are
naturally present in addition to the bacteria’s other DNA. They contain only a few
of the thousands of genes that are present in a bacterium genome and can be copied
many times in a single bacterial cell. In library construction some of the enzyme cut
DNA pieces will combine with the cut plasmids and form recombinant DNA (or
DNA in a ’new combination’).
The recombinant plasmids are then transferred into bacteria using either
electroporation or heat shock. Electroporation uses mild pulses of electricity to
disrupt the cell walls of the bacterium and create small holes. The plasmids are small
enough to pass through the holes into the cell. Heat shock works in a similar fashion.
However, rather than using electricity to create holes in the bacterium, it is done by
alternating the temperature between hot and cold.

After undergoing electroporation, transformation or heat shock, the bacteria are

plated out onto petri dishes. If each bacteria contains a different gene-size segment
of the extracted DNA, it may take thousands of bacteria, and thus hundreds of plates,
to contain all of the segments.

When the recombinant plasmid is inside the bacterium, the bacterium is fooled into
thinking the new DNA is its own and begins replicating it. The plasmid can be
replicated independently of the bacterium’s own DNA. The bacteria also multiply
into colonies, with each colony coming from a cell that has been transformed with a
different piece of recombinant DNA. The total of all of these colonies makes up the
gene library. The scientist must then locate the colony that contains the gene of
interest.

Screening the Library

The gene cloner must then screen the library in order to discover which bacterial
colony is making copies of the one gene they are interested in. Library screening
identifies colonies, which have that particular gene. Screening can be based on
 Detecting the DNAsequence of the cloned gene,
 Detecting a protein that the gene encodes,
 Or the use of linked DNA markers.
Therefore before library screening can be done, the scientist must know either the
DNA sequence of the gene, or a very similar gene, the protein that the gene produces,
or a DNA marker that has been mapped very close to the gene.
When the bacteria multiply and replicate the recombinant DNA, the number of gene
copies also increases, making gene or protein detection easier. When the bacteria
colony containing the desired gene is located, the bacteria can be propagated to make
millions of copies of the recombinant plasmid that contains the gene. The plasmids
can be extracted for the next steps of genetic engineering, gene modification, and
transformation.