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From the double helix
to the genome
Bioinformatics
Genetic engineering has boosted the development of
different branches of science such as biochemistry,
genetics and molecular biology.
The computer proved an indispensable tool in managing,
processing and connecting the huge amount of data
that emerged from genetic techniques.
BIOINFORMATICS
From the double helix to the genome> Bioinformatics
Recombinant DNA
Bioengineering
techniques for the manipulation and
analysis of DNA outside the cell.
recombinant DNA technology makes it possible
to isolate and cut short sequences of
DNA before transferring and inserting them into
the genome of other cells in order to
modify the expression of one or more genes.
Targeted transfer of DNA may also take place between
the genetic material belonging to different species.
From the double helix to the genome > Recombinant DNA
Cloning
In 1973, Stanley Cohen and Herbert Boyer began to
develop a new technique called cloning and managed to
create the first “genetically modified” organism in
history.
From the double helix to the genome > Cloning
Technique of cloning
It is possible:
• to insert the gene region of interest into a plasmid
• to produce large quantities of the cloned region through the
transfer of the vector into bacteria
• to select only the bacteria that contain the recombinant
vector a gene for resistance to an antibiotic is used.
From the double helix to the genome >Technique of cloning
Enzymes and restriction sites
Restriction enzymes are proteins capable of
cutting the DNA at precise nucleotide
sequences, called restriction sites.
Restriction enzymes
• are produced by bacteria to protect themselves from
viruses that parasitize them, cutting their DNA.
• are able to act on the foreign DNA only.
From the double helix to the genome > Enzymes and restriction sites
Sticky ends
A restriction site is a palindromic sequence of 4, 6 or 8
pairs of nitrogenous bases.
The majority of restriction enzymes work by making a
staggered cut at the level of their
specific palindrome sequences, forming sticky ends.
From the double helix to the genome > Enzymes and restriction sites
PCR: the polymerase chain reaction
Starting in 1985, this technique was designed allowing the
in vitro amplification of DNA.
PCR is a technique that, using a DNA polymerase,
allows the rapid production of millions of identical
copies of a DNA region, starting from extremely small
quantities of material.
The nucleotide sequences that surround it
perfectly. Is the ‘moulds’ for the in vitro synthesis
of the triggers from which the duplication reaction
of the target region will then take place.
The proteins and nucleic acids > PCR: the polymerase chain reaction
The primers
The primers, that initiate the reaction, are two
sequences of 17-30 nucleotides, complementary
to the sequences of the target DNA that constitute,
respectively, the beginning of a filament and the end of it
on the opposite strand.
The proteins and nucleic acids > The primers
The thermal cycler
The replication reaction of the DNA region of interest takes
place in a machine called a “thermal cycler” and regulates
the succession of amplification cycles during which it
alternates 3 different temperatures:
•94 ˚C: denaturation of double-stranded DNA template into
two single strands through heating;
•30-65 ˚C: annealing of the primers to the sequences of
single-stranded DNA
•65-75 ˚C: primer extension by addition of
deoxyribonucleotides in the 5’→3’ direction carried out by the
DNA polymerase.
The proteins and nucleic acids > The thermal cycler
Gene sequencing
The genome as the ‘instruction manual’ that describes the
functioning of all living organisms.
MAP OF THE GENES
GENETIC MAPPING
the relative distance
between genes
The proteins and nucleic acids > Gene sequencing
PHYSICAL MAPPING
based on the techniques
used in molecular biology to
build maps that show the
physical location of the genes
The Sanger method
The turning point for DNA sequencing was determined by
the possibility of using DNA polymerase outside the cellular
environment.
1975, The Sanger method.
A terminator is a molecule of dideoxyribonucleotide
triphosphate (dNTP), that is to say a
molecule of deoxyribonucleotide triphosphate (dNTP) in
which, in the molecule of ribose,
only one –OH group has been replaced
with a hydrogen atom.
The proteins and nucleic acids > DNA sequencing with terminators
Electrophoresis of DNA fragments
The Sanger method of DNA sequencing provides a
mixture of DNA fragments which must be separated
The Gel Electrophoresis Technique
Gel electrophoresis is a method that exploits the
chemical characteristics of molecules
(nucleic acids or proteins) to separate them and
identify them thanks to their differing
behaviours under the action of an electric field.
The proteins and nucleic acids > Electrophoresis of DNA fragments
An incomplete genome: Dark DNA
The Human Genome Project
The goal (1987) was to
determine the nucleotide sequence
of human genetic makeup,
identifying and mapping the genes
that make it up - a goal that was
achieved in 2003.
HOWEVER
The identification of the function of each gene
was still a long way off.
The proteins and nucleic acids > An incomplete genome: Dark DNA
Outcomes and evidences
Only 2% of the human genome
consists of genes and the
remaining part which normally
does not encode, was hastily
termed “junk DNA”.
At the end of the project,
however, they had identified
‘only’ 20- 30,000 genes, instead
of the 100,000 expected.
The complexity of an
organism does not only
depend on the number of
genes contained in its DNA.
The proteins and nucleic acids > Outcomes and evidences
DNA is not
the sole repository of
genetic information.
Non-coding DNA may
still play a role in
gene expression,
backup or in
regulation.
It serve to ‘switch’
genes ‘on’ or ‘off’,
thus triggering, or
blocking.
The Encode project
Deeper study of the human genome and to obtain a map,
which identifies 4 million new stretches of DNA that, in
effect, act as (on-off) switches in the regulation of gene
expression.
The proteins and nucleic acids > The Encode project