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Objectives: - We want to prove the existence of a genetic inheritance in all the alive beings - We search to know if the genetic inheritance is identical at all the species. - We search to know if the genetic inheritance is transmissible in experiments of a species to another. Does there exist a genetic material in all the alive beings? We wants to extract from the DNA of different cellular species such as: - Plant species : the onion - Animal species eukaryote* pluricellular : saliva - Animal species eukaryote* pluricellular : liver of mouse - Animal species prokaryote** : bacterium *: cell :containing a core **: cell: not having a core Material DNA human DNA of onion DNA of liver DNA of bacterium - Saliva -Onion - test tube of 50ml - Plug (TE*+SDS+Na Cl) - A centrifuge -Alcohol (ethanol) -A lump of liver (2g) - a Potter - Tube of 50 ml - Plug (TE*+SDS+NaCl ) -Alcohol (ethanol) -Bacterium - a centrifuge - Streamroom - culture medium - Plug ( TE*+ SDS+NaCl) -Alcohol (ethanol) - test tube of 15 ml - Plug ( TE*+SDS+Na Cl) - Alcohol (ethanol) *: Plug Tris-EDTA, pH 8 Procedure of extraction of DNA of liver • LIVER: • Chop a liver • Put in a potter and crush • Put in a plug (SDS+NaCl+ TE) to make burst the cells. • Centrifuge • To eliminate the supernatant • Add the ethanol • Recover the ball supposed to be DNA • Procedure of extraction of DNA of bacterium BACTERIUM: • Setting in culture of the bacterium • Put in a tube eppendorf • Centrifuge • Eliminate the culture medium • Put in the plug (SDS+NaCl+TE) • Agitate after add ethanol • Put in the refrigerator • Recover the ball supposed to be DNA Procedure of extraction of DNA of human saliva HUMAN SALIVA: • Collect human saliva • Pour in a tube of 15ml • Pour the plug (SDS +NaCl+TE) in this tube • Pour ethanol • Let rest • Recover the ball supposed to be DNA Ball of DNA Procedure of extraction of DNA of onion • ONION • Chop the onion • Put in a tube of 50mL • Put in the plug (SDS+NaCl+TE) • Centrifuge • Add the ethanol • recover the ball supposed to be DNA First objective’s conclusion : We observe a ball, that supposing to us to be DNA, in the four tubes of the various cellular species. If that is checked, we can highlight a genetic inheritance in the alive beings tested. Test of characterization of the DNA Thanks to the composition of the DNA that we know, we carried out, in class, an experiment aiming at proving the presence of the various elements composing the DNA. A coloring specific to the DNA consolidated us in our idea that the collected ball was well of the DNA. Is the genetic inheritance identical between species? • One treats the DNA with the protein K "to degrade" proteins related to the DNA. A fraction of this DNA was treated with enzymes of restriction. Analyze by electrophoresis on GEL of agar of DNAs extracted 1 2 3 4 5 6 7 8 9 1 MARKER OF SIZE 2 DNA OF LIVER 3 DNA OF BACTERIE 4 ONION DNA 5 DNA OF SALIVA 6 DNA OF LIVER DIGESTED 7 DNA OF BACTERIE DIGESTED 8 ONION DNA DIGESTED 9 DNA OF SALIVA DIGESTED Second objective’s conclusion: • One observed thanks to the technique of electrophoresis that between the species tested the genetic inheritance was not identical because the migrations of the DNA of each species were not the same ones. Can one transmit the genetic inheritance of one species to the other? • MATERIALS: • Bacteria • - DNA coding the GFP (Fluorescent Green Protein) taken with the jellyfish • - box of Petri containing agar • - Plasmid (circular DNA) • - Starters • - Taq DNA Polymerase • - DNTP • - Plug for PCR • - Thermocyclor • - Tubes of PCR Technique of the PCR Séquence cible GFP Denaturising of the DNA Cycle 1 Hybridizing Elongation Cycle 2 Cycle 3 We have to carry out 29 cycles TRANSFER OF THE GENE OF GFP RESULTING FROM MEDUSE TO THE BACTERIE (Escherichia Coli) GFP Plasmid Bacteria Transformation (introduction of the plasmid) Transformed bacterium (GMO) Obtaining genetically modified bacteria: fluorescence GFP Bacteria (control) Genetically modified bacteria General conclusion We have to carry out three experiments aiming at proving the existence of the DNA in all the species. We have deduce the existence from the DNA, its authenticity between the species and the possibility of transmission of the genetic information from one species to another in laboratory. Au vue des résultats de nos expériences, nous pouvons déduire qu’ il existe effectivement un patrimoine génétique chez tous les êtres vivants testés.