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 1/3 of the prize 1/3 of the prize 1/3 of the prize Switzerland USA USA

Biozentrum der Universität Johns Hopkins University Johns Hopkins University Basel, Switzerland School of Medicine School of Medicine

Baltimore, MD, USA Baltimore, MD, USA

b. 1929 b. 1928 b. 1931d. 1999

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 non-reproductive 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 insertsBAC = 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 Howard Martin Temin

1/3 of the prize 1/3 of the prize 1/3 of the prize USA USA USA

Massachusetts Institute Imperial Cancer Research University of Wisconsin

of Technology (MIT) Fund Laboratory Madison, WI, USA

Cambridge, MA, USA London, United Kingdom

b. 1938 b. 1914 b. 1934

(in Catanzaro, Italy)

Genoteca di cDNA

I cDNA posono espressi in cellule batteriche se si usa un promotorebatterico

Identificazione di un clone specifico in una genoteca classicaIbridizzazione con sonde di DNA :

Mediante anticorpiPer evidenziare la Proteina prodotta dal Vettore d’espressione.

Polymarase Chain ReactionPCR

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, site-directed mutagenesis and its development for protein studies"

Michael Smith1/2 of the prizeUniversity of British ColumbiaVancouver, Canada

b. 1932(in Blackpool, United Kingdom)d. 2000

T > 950C

Tm primers

T DNA pol.T > 950C