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UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia 2.1. ADN recombinant, enzims de restricció Formació d’ADN recombinant. Utilització d’enzims de restricció. Lloc de restricció, és le punt on l’enzim pot tallar l’ADN Fragment de restricció. Figure 20.3-3 Restriction site 5 3 GAATTC CTTAAG DNA 5 3 1 Restriction enzyme cuts sugar-phosphate backbones. 5 3 5 3 5 Sticky 3 3 5 end 5 2 DNA fragment added 3 3 5 from another molecule cut by same enzyme. Base pairing occurs. 5 3 5 3 3 DNA ligase 3 5 G AATT C C TTAA G G AATT C C TTAA G 53 5 3 3 5 One possible combination seals strands 5 3 3 Recombinant DNA molecule 5 UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia 2.2. Clonació d’un gen eucariont LE 20-4_3 Bacterial cell Isolate plasmid DNA and human DNA. Clonació usant plàsmids bacterians lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Cut both DNA samples with the same restriction enzyme. Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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 nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone LE 20-5 Hibridació amb sonda d’àcid nucleic Master plate Filter Radioactive single-stranded DNA Solution containing probe Master plate Probe DNA Colonies containing gene of interest Gene of interest Single-stranded DNA from cell Film 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. LE 20-6 or Bacterial clones Recombinant plasmids Plasmid library Foreign genome cut up with restriction enzyme Recombinant phage DNA Phage clones Phage library UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia 2.4. Amplificació de l’ADN: reacció en cadena de la polimerasa (PCR) Aquesta tècnica s’usa quan es disposa de quantitats molt petites d’ADN. En poc temps s’aconsegueixen milions de còpies. Aplicació a l’estudi de l’ADN dels neandertals 5 3 Target sequence Genomic DNA Denaturation: Heat briefly to separate DNA strands Cycle 1 yields 2 molecules Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Extension: DNA polymerase adds nucleotides to the 3 end of each primer Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence 3 5 5 3 3 5 Primers New nucleotides