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Chp 20: DNA Technology and Genomics Activity 20A LE 20-2 Bacterium Gene inserted into plasmid Cloning Bacterial chromosome Cell containing gene of interest Plasmid Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed by gene of interest Copies of gene Basic research on gene Gene for pest resistance inserted into plants Protein harvested Basic research and various applications Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth LE 20-3 Restriction site Recombinant DNA DNA 5 3 3 5 Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme RJ animation One possible combination DNA ligase seals the strands. Activity: Restriction Enzymes Recombinant DNA molecule LE 20-4_3 Bacterial cell Cloning using a bacterial plasmid Isolate plasmid DNA and human DNA. 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 Activity: Cloning a gene in bacteria Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone RJ animation LE 20-5 Nucleic Acid Hybridization Master plate Filter Master plate Probe DNA Radioactive single-stranded DNA Solution containing probe 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 Genomic Library or Bacterial clones Foreign genome cut up with restriction enzyme Recombinant plasmids Recombinant phage DNA Plasmid library Phage clones Phage library RJ animation cDNA LE 20-7 5 3 PCR 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 RJ animation New nucleotides 3D video (amplification techniques) LE 20-8 Gel Electrophoresis Cathode Power source Mixture of DNA molecules of different sizes Longer molecules Shorter molecules Gel Glass plates Anode Activity: Gel Electrophoresis Southern Blotting Restriction fragments DNA + restriction enzyme I II III Heavy weight Nitrocellulose paper (blot) Gel Sponge I Normal -globin allele II Sickle-cell III Heterozygote allele Preparation of restriction fragments. Radioactively labeled probe for -globin gene is added to solution in a plastic bag I II III Paper towels Alkaline solution Gel electrophoresis. Probe hydrogenbonds to fragments containing normal or mutant -globin Blotting. I II Fragment from sickle-cell -globin allele Paper blot Hybridization with radioactive probe. III Film over paper blot Fragment from normal -globin allele Autoradiography. LE 20-9 Normal -globin allele Restriction Fragment Analysis 175 bp Ddel 201 bp Ddel Large fragment Ddel Ddel Sickle-cell mutant -globin allele 376 bp Ddel Large fragment Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles RJ animation LE 20-11 Chromosome bands Cytogenetic map Mapping Genes located by FISH Genetic (linkage) mapping Genetic markers Physical mapping Overlapping fragments DNA sequencing LE 20-12 DNA (template strand) 5 Primer 3 Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) Dideoxymethod (Sanger) DNA sequencing 5 DNA polymerase 3 5 DNA (template strand) Labeled strands 3 Direction of movement of strands Laser Detector 3 LE 20-13 Cut the DNA from many copies of an entire chromosome into overlapping fragments short enough for sequencing Clone the fragments in plasmid or phage vectors Sequence each fragment Order the sequences into one overall sequence with computer software Table 20-1 LE 20-14 DNA Microarray Assay Tissue sample Isolate mRNA. Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of singlestranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. mRNA molecules Labeled cDNA molecules (single strands) DNA microarray Size of an actual DNA microarray with all the genes of yeast (6,400 spots) Figure 20-1 DNA Arrays & Gene Chips RJ animation LE 20-15 RFLP marker analysis RFLP marker DNA Restriction sites Disease-causing allele Normal allele LE 20-16 Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Retrovirus capsid Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Inject engineered cells into patient. Bone marrow LE 20-17 Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V) Activity: DNA Fingerprinting Video: DNA Forensics Figure 20-18 Insulin & Genentech Transgenics video Figure 20.20 “Golden” rice contrasted with ordinary rice Activity: Golden Rice LE 20-19 Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid RJ animation Plant with new trait