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Chapter 20

Chapter 20. DNA Technology and Genomics. DNA Cloning. Tools and techniques: Making Recombinant DNA using restriction enzymes Cloning vector – the bacterial plasmid Nucleic Acid Probe Hybridization – identifying clones Genomic libraries PCR. Cell containing gene of interest. Bacterium.

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Chapter 20

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  1. Chapter 20 DNA Technology and Genomics

  2. DNA Cloning • Tools and techniques: • Making Recombinant DNA using restriction enzymes • Cloning vector – the bacterial plasmid • Nucleic Acid Probe Hybridization – identifying clones • Genomic libraries • PCR

  3. Cell containing gene of interest Bacterium Gene inserted into plasmid LE 20-2 Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

  4. Cell containing gene of interest Bacterium LE 20-2a Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Recombinant bacterium

  5. Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest LE 20-2b Protein expressed by gene of interest Gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

  6. Restriction site 5¢ 3¢ DNA 3¢ 5¢ LE 20-3 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 One possible combination DNA ligase seals the strands. Recombinant DNA molecule

  7. Bacterial cell lacZ gene (lactose breakdown) Human cell LE 20-4_1 Isolate plasmid DNA and human DNA. 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

  8. Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. LE 20-4_2 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

  9. lacZ gene (lactose breakdown) Bacterial cell Human cell Isolate plasmid DNA and human DNA. Restriction site LE 20-4_3 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

  10. Colonies containing gene of interest LE 20-5 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.

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

  12. 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ LE 20-7 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

  13. Restriction Fragment Analysis • Gel electrophoresis • Southern blotting • Restriction Fragment Length Differences - RFLPs

  14. Mixture of DNA molecules of differ- ent sizes Longer molecules LE 20-8 Cathode Shorter molecules Power source Gel Glass plates Anode

  15. Normal b-globin allele 175 bp 201 bp Large fragment LE 20-9 Ddel Ddel Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Large fragment Ddel 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

  16. Normal b-globin allele LE 20-9a 175 bp 201 bp Large fragment Ddel Ddel Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Large fragment Ddel Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene

  17. Normal allele Sickle-cell allele LE 20-9b Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

  18. Heavy weight Restriction fragments DNA + restriction enzyme Nitrocellulose paper (blot) I I I Gel LE 20-10 Sponge Paper towels I Normal -globin allele I Sickle-cell allele I Heterozygote Alkaline solution Preparation of restriction fragments. Gel electrophoresis. Blotting. Probe hydrogen- bonds to fragments containing normal or mutant -globin I I I Radioactively labeled probe for -globin gene is added to solution in a plastic bag I I I Fragment from sickle-cell -globin allele Film over paper blot Fragment from normal -globin allele Paper blot Hybridization with radioactive probe. Autoradiography.

  19. Mapping Genes • Human Genome Project • Steps of mapping the genome • Genetic mapping • Physical Mapping • DNA sequencing • Sanger (Fig. 20.12) • Venter (Fig. 20.13)

  20. Chromosome bands Cytogenetic map Genes located by FISH LE 20-11 Genetic (linkage) mapping Genetic markers Physical mapping Overlapping fragments DNA sequencing

  21. DNA (template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) 3¢ 5¢ 5¢ LE 20-12 DNA polymerase 3¢ DNA (template strand) Labeled strands 3¢ 5¢ 3¢ Direction of movement of strands Laser Detector

  22. Cut the DNA from many copies of an entire chromosome into overlapping frag-ments short enough for sequencing LE 20-13 Clone the fragments in plasmid or phage vectors Sequence each fragment Order the sequences into one overall sequence with computer software

  23. Genomics - Applications • Determining Gene function • In vitro mutagenesis • RNA interference • Gene expression studies • DNA microarray assays • Comparing genomes • Proteonomics • SNPs

  24. Tissue sample Isolate mRNA. LE 20-14 mRNA molecules Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded 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. Labeled cDNA molecules (single strands) DNA microarray Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Size of an actual DNA microarray with all the genes of yeast (6,400 spots)

  25. Practical Applications • Medical • Diagnosis of disease • RT-PCR • Gene therapy • Pharmaceuticals • Forensics • Agriculture • Environmental

  26. LE 20-15 RFLP marker DNA Disease-causing allele Restriction sites Normal allele

  27. Cloned gene Insert RNA version of normal allele into retrovirus. LE 20-16 Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow Inject engineered cells into patient.

  28. Agrobacterium tumefaciens Ti plasmid LE 20-19 Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid Plant with new trait

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