Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

Isolamento dei geni

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

Patologie causate da difetti geneticiPatologia Difetto Incidenza Cellule bersaglioGeneticaImmunodeficienza Adenosina deaminasi Rara Cellule del midollo osseocombinata oppure linfociti T(SCID/ADA)Emofilia A Mancanza di Fattore VIII 1:10,000 maschi Fegato, muscolo, fibroblasti

“ B Mancanza di Fattore IX 1:30,000 maschiIpercolestrolemia Mancanza del recettore per la 1:1 milione Fegato

low-density Lipoprotein (LDL)Fibrosi cistica CFTR 1:3,000 caucasici Vie aereeEmoglobinopatie: Difetti nei geni della 1:600 in alcuni Cellule del midollo osseoTalassemie/SCA α- o β-globina gruppi etnici precursori dei globuli rossiDistrofia muscolare Distrofina 1: 3,500 maschi Fibre muscolari striatedi DuchenneEnfisema ereditario Mancanza di of α1-antitripsina 1:3,500 Polmoni o fegatoAquisitaCancro Varie cause 1milione/anno in USA Vari tipi cellulariMalattie neurologiche Parkinson, Alzheimer Parkinson: 1 milione Neuroni, cellule della glia

Alzheimer: 4 milioni o di Schwann pazienti negli USA

Cardivascolari Restenosi, aterosclerosi 13 milioni negli USA Arterie, cellule endotelialiMalattie infettive AIDS, epatite C Pazienti in aumento Cellule T, fegato, macrofagi

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations– the discovery of sequence-specific endonucleases

provided the necessary scissors• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined to

plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA fromthe test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations– the discovery of sequence-specific endonucleases

provided the necessary scissors• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined to

plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA fromthe test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.

isolation

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations– the discovery of sequence-specific endonucleases

provided the necessary scissors• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined to

plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA fromthe test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations– the discovery of sequence-specific endonucleases

provided the necessary scissors• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined to

plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA fromthe test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

Synthesis of recombinant DNA molecules

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

Synthesis of recombinant DNA molecules

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations– the discovery of sequence-specific endonucleases

provided the necessary scissors• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined to

plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA fromthe test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations– the discovery of sequence-specific endonucleases

provided the necessary scissors• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined to

plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA fromthe test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations– the discovery of sequence-specific endonucleases

provided the necessary scissors• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined to

plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA fromthe test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations– the discovery of sequence-specific endonucleases

provided the necessary scissors• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined to

plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA fromthe test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.

Prof. Savino; dispense di Biologia Molecolare, Corso di Laurea in Biotecnologie

DNA cloningCloning a segment of DNA entails five general procedures:

• a method for cutting DNA at precise locations

– the discovery of sequence-specific endonucleases provided the necessary scissors

• a method for joining two fragments covalently

– DNA ligase can do this• selection of small molecules capable of DNA

replication– segments of DNA to be cloned can be joined

to plasmids or viral DNA (cloning vectors); these composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs

• a method for moving the recombinant DNA from the test tube into a host cell that can provide the enzymatic machinery for DNA replication;

• methods to select or identify those host cells that contain recombinant DNA.