UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia 2.1. ADN recombinant , e nzims de restricció

1. UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia 2.1. ADN recombinant, enzims de restricció Formaciód’ADNrecombinant. Utilitzaciód’enzims de restricció. Lloc de restricció, és le puntonl’enzimpot tallar l’ADN Fragment de restricció.

2. 1 2 3 Figure 20.3-3 Restriction site 5 3 GAATTC DNA CTTAAG 5 3 Restriction enzymecuts sugar-phosphatebackbones. 5 3 3 5 AATTC G CTTAA G 5 Sticky end 3 5 3 5 3 AATTC G G CTTAA DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 3 5 5 3 5 3 5 3 G AATT C G AATT C C TTAA G G C TTAA 3 5 3 5 5 3 One possible combination DNA ligaseseals strands 5 3 3 5 Recombinant DNA molecule

3. UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia 2.2. Clonaciód’un gen eucariont

4. LE 20-4_3 lacZ gene (lactose breakdown) Bacterial cell Human cell Isolate plasmid DNA and human DNA. Clonacióusant plàsmidsbacterians Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone

5. LE 20-5 Hibridacióamb sonda d’àcidnucleic Colonies containing gene of interest Master plate Master plate Probe DNA Radioactive single-stranded DNA Solution containing probe Gene of interest Film Single-stranded DNA from cell Filter Filter lifted and flipped over Hybridization on filter A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

6. LE 20-6 Foreign genome cut up with restriction enzyme or Bacterial clones Recombinant plasmids Phage clones Recombinant phage DNA Plasmid library Phage library

7. UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia 2.4. Amplificació de l’ADN: reacció en cadena de la polimerasa (PCR) Aquestatècnicas’usaquan es disposa de quantitatsmoltpetitesd’ADN. En poctempss’aconsegueixenmilions de còpies. Aplicació a l’estudi de l’ADNdelsneandertals

8. 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ 5¢ 3¢ Denaturation: Heat briefly to separate DNA strands 3¢ 5¢ Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Cycle 1 yields 2 molecules Primers Extension: DNA polymerase adds nucleotides to the 3¢ end of each primer New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence