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The Nobel Prize in Physiology or Medicine 1978
"for the discovery of restriction enzymes and their
application to problems of molecular genetics"
Werner Arber
Daniel Nathans
Hamilton O. Smith
Biozentrum der Universität
Basel, Switzerland
Johns Hopkins University
School of Medicine
Baltimore, MD, USA
Johns Hopkins University
School of Medicine
Baltimore, MD, USA
b. 1929
b. 1928
d. 1999
b. 1931
1/3 of the prize
Switzerland
1/3 of the prize
USA
1/3 of the prize
USA
RESTRICTION ENZYMES
• function : to “restrict” infection by phages
• endonuclease site specific : each enzyme recognizes either
4 or 6 or 8 base pairs and cuts in correspondence of the
recognized site
Rescriction enzymes recognise palindromic sequences
RESTRICTION ENZYMES
•Endonuclease site specific : recognizes 4-6-8 base pairs and cut
in correspondence of the recognized site.
cleavage site symmetry
Auto protection from cleavage
Restriction maps
DNA ligase
Cloning is the process of creating an
identical copy of an original organism or
thing. A cloning in the biological sense, is
a molecule, a single cell (like bacteria or
yeast , eukariotes etc.) or a multi-cellular
organism that has been directly copied
from and is therefore genetically identical
to another living organism.
Molecular cloning refers to the
procedure isolating a DNA sequence of
interest and obtaining multiple copies of
it in an organism. Cloning is frequently
employed to amplify DNA fragments
containing genes, an essential step in
their subsequent analysis. Cloning of
any DNA sequence involves the
following four steps: amplification,
ligation, transfection, and
screening/selection.
Cloning a cell means to derive a
(clonal) population of cells from a
single cell. This is an important in
vitro procedure when the
expansion of a single cell with
certain characteristics is desired,
for example in the production of
gene-targeted ES cells. Most
individuals began as a single cell
and are therefore the result of
clonal expansion in vivo.
Cloning an organism means to create a new organism with the same
genetic information as a cell from an existing one(identical). It is an
asexual method of reproduction, where fertilization or inter-gamete
contact does not take place. This form of reproduction is common
among simple organisms such as bacteria.
Dolly (1996) was the first mammal to
have been successfully cloned from an
adult cell. She was cloned at the Roslin
Institute in Scotland and lived there until
her death when she was 6. The name
"Dolly" in honor of Dolly Parton,
because it was a mammary cell that
was cloned. The technique that was
made famous by her birth is somatic cell
nuclear transfer, in which a nonreproductive cell containing a nucleus is
placed in a de-nucleated ovum (which
then develops into a fetus).
1972 (October) Paul Berg and
co-workers create the first recombinant
DNA molecule (PNAS).
Trasformazione = inserimento un DNA
in un batterio o in un lievito
Trasfezione = inserimento di un DNA
In una cellula di eucarioti
DNA viral vectors lamda (l) phage
Advantages:
• bigger insert with respect to plasmids (~20kb)
• bacterial cell lysis (expression vector)
Cloning DNA fragments 32-45 kb with cosmids
Cloning DNA fragments 32-45 kb with cosmids
YAC = yeast artificial chromosomes for 600 kb to 2 Mb inserts
BAC = bacterial artificial chromosomes to insert ~ 300 kb.
Genoteca genomica = collezione del DNA di un genoma
di un organismo in frammenti clonati
Quanti cloni indipendenti sono necessari
per rappresentare un intero genoma?
Un “array” ordinato permette
di identificare un gene
dal suo posizionamento nell
“array”.
The Nobel Prize in Physiology or Medicine 1975
"for their discoveries concerning the interaction between tumour viruses and
the genetic material of the cell"
David Baltimore
Renato Dulbecco
1/3 of the prize
USA
1/3 of the prize
USA
Massachusetts Institute
Imperial Cancer Research
University of Wisconsin
of Technology (MIT)
Fund Laboratory
Madison, WI, USA
Cambridge, MA, USA
Kingdom
b. 1938
Howard Martin
Temin
1/3 of the prize
USA
London, United
b. 1914
(in Catanzaro, Italy)
b. 1934
Genoteca di cDNA
I cDNA posono espressi in cellule batteriche se si usa un promotore
batterico
Identificazione di un clone specifico in una genoteca classica
Ibridizzazione con sonde di DNA :
Mediante anticorpi
Per evidenziare la
Proteina prodotta dal
Vettore d’espressione.
Polymarase Chain Reaction
PCR
The Nobel Prize in Chemistry 1993
"for contributions to the developments of methods within DNA-based chemistry"
"for his invention of the
polymerase chain reaction
(PCR) method"
Kary B. Mullis
1/2 of the prize
La Jolla, CA, USA
b. 1944
"for his fundamental contributions
to the establishment of
oligonucleotide-based, sitedirected mutagenesis and its
development for protein studies"
Michael Smith
1/2 of the prize
University of British Columbia
Vancouver, Canada
b. 1932
(in Blackpool, United Kingdom)
d. 2000
T > 950C
Tm primers
T > 950C
T DNA pol.