DNA Technology and Genomics Chapter 12 DNA Technology and Genomics

Figure 12.1 An overview of gene cloning (layer 1) Plasmid Bacterium Bacterial chromosome Plasmid isolated Recombinant DNA (plasmid) Gene inserted into plasmid DNA isolated Cell containing gene of interest DNA Gene of interest Plasmid put into bacterial cell Recombinant bacterium Cell multiplies with gene of interest Copies of protein Protein used to make snow form at higher temperature Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy 1 2 3 4 5

Figure 12.1 An overview of gene cloning (layer 2) Plasmid Bacterium Bacterial chromosome Plasmid isolated Recombinant DNA (plasmid) Gene inserted into plasmid DNA isolated Cell containing gene of interest DNA Gene of interest Plasmid put into bacterial cell Recombinant bacterium Cell multiplies with gene of interest Copies of protein Protein used to make snow form at higher temperature Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy 1 2 3 4 5

Figure 12.1 An overview of gene cloning (layer 3) Plasmid Bacterium Bacterial chromosome Plasmid isolated Recombinant DNA (plasmid) Gene inserted into plasmid DNA isolated Cell containing gene of interest DNA Gene of interest Plasmid put into bacterial cell Recombinant bacterium Cell multiplies with gene of interest Copies of protein Protein used to make snow form at higher temperature Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy 1 2 3 4 5

Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase Restriction enzyme recognition sequence G A A T T C C T T A A G DNA 1 2 3 4 C T T A A A AT TC G Addition of a DNA fragment from another source Two (or more) fragments stick together by base-pairing G A AT T C C T TA A G 5 DNA ligase Pastes the strand Restriction enzyme cuts the DNA into fragments Recombinant DNA molecule Sticky end

Figure 12.3 Cloning a gene in a bacterial plasmid (layer 1) E.coli Plasmid Isolate DNA from two sources Cut both DNAs with the same restriction enzyme Human cell DNA 2 1 Gene V Sticky ends

Figure 12.3 Cloning a gene in a bacterial plasmid (layer 2) E.coli Plasmid Isolate DNA from two sources Cut both DNAs with the same restriction enzyme Human cell DNA 2 1 3 4 Gene V Sticky ends Mix the DNAs; they join by base-pairing Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid

Figure 12.3 Cloning a gene in a bacterial plasmid (layer 3) E.coli Plasmid Isolate DNA from two sources Cut both DNAs with the same restriction enzyme Human cell DNA 2 1 3 4 5 6 Gene V Sticky ends Mix the DNAs; they join by base-pairing Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid Put plasmid into bacterium by transformation Recombinant bacterium Clone the bacterium Bacterial clone carrying many copies of the human gene

12.3 Cloning a gene

Figure 12.7A Human insulin produced by bacteria

Figure 12.18B Transgenic pigs

Figure 12.6 “Pharm” animals that produce a human protein

gene within plant chromosome Figure 12.18A Using the Ti plasmid as a vector for genetically engineering plants Agrobacterium tumefaciens DNA containing gene for desired trait Ti plasmid 1 2 3 Insertion of gene into plasmid using restriction enzyme and DNA ligase Recombinant Ti plasmid Introduction into plant cells in culture Regeneration of plant Plant with new trait T DNA carrying new gene within plant chromosome Plant cell T DNA Restriction site

Figure 12.19B Pollen might transfer genes from genetically engineered crop plants to wild relatives nearby

Table 12.6 Some protein products of recombinant DNA technology

Figure 12.14 DNA amplification by PCR 8 Initial DNA segment Number of DNA molecules

Figure 12.11A Restriction site differences between two homologous samples of DNA Crime scene Suspect w x y z Cut DNA from chromosomes C G A T

Figure 12.10 Gel electrophoresis of DNA + – Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter Completed gel

Figure 12.12A DNA fingerprints from a murder case Defendant’s blood Blood from defendant’s clothes Victim’s

Figure 12.12B DNA data for forensic use

Unnumbered Figure p. 231 Investigator at one of the crime scenes (above), Narborough, England (left)

Unnumbered Figure p. 230

Figure 12.8 How a DNA probe tags a gene by base pairing Radioactive probe (DNA) Single-stranded DNA Mix with single- stranded DNA from various bacterial (or phage) clones Base pairing indicates the gene of interest A T C C G A A T G C G C T T A T C G A G C C T T A T G C A T A G G T A G G C T A A

Figure 12.11B Gel electrophoresis of restriction fragments – + Longer fragments shorter x w y z 1 2

Figure 12.11C The use of restriction fragment analysis to detect a harmful allele (layer 1) preparation Gel electrophoresis I II III Restriction fragments

Figure 12.11C The use of restriction fragment analysis to detect a harmful allele (layer 2) 3 4 Restriction fragment preparation Gel electrophoresis Blotting Radioactive probe I II III Restriction fragments Filter paper Probe Radioactive, single- stranded DNA (probe)

Figure 12.11C The use of restriction fragment analysis to detect a harmful allele (layer 3) 4 5 Restriction fragment preparation Gel electrophoresis Blotting Radioactive probe Detection of radioactivity (autoadiography) I II III Restriction fragments Filter paper Probe Radioactive, single- stranded DNA (probe) Film

Figure 12.7B Equipment used in the production of a vaccine against hepatitis B

Figure 12.13 One type of gene therapy procedure 4 3 2 1 Cloned gene (normal allele) Insert normal gene into virus Infect bone marrow cell with virus Viral DNA inserts into chromosome Inject cells into patient Bone marrow Bone marrow cell from patient Viral nucleic acid Retrovirus

Figure 12.9 DNA microarray 1 4 3 2 mRNA isolated Reverse transcriptase and fluorescent DNA nucleotides cDNA made from mRNA Unbound cDNA rinsed away cDNA applied to wells DNA microarray Each well contains DNA from a particular gene Actual size (6,400 genes) Nonfluorescent spot Fluorescent cDNA DNA of an expressed gene unexpressed gene

Figure 12.15 DNA sequencing

Table 12.17 Some important completed genomes

Figure 12.19A A maximum-security laboratory at the Pasteur Institute in Paris

Figure 12.20 Eric Lander

Unnumbered Figure p. 250 Bacterial clone Cut Bacterium DNA fragments Plasmids Recombinant plasmids Bacterium Bacterial clone bacteria Genomic library

Unnumbered Figure p. 250 – + Mixture of DNA fragments Longer A “band” is a collection fragments of one particular length DNA attracted to + pole due to PO4– groups Shorter move faster Longer move slower Power source – +

Unnumbered Figure p. 250 Unknown DNA amplified (a) via Bacterial sample Suspect sample for comparison Bacterial plasmids treated with fragments sorted by size via (b) (c) (d) (e) (f) (g) Visualizing and comparing fragments is a method called Recombinant plasmids are inserted into bacteria Add Particular sequence highlighted are copied via Collection is called a

Figure 12.4 Genomic libraries Recombinant plasmid Genome cut up with restriction enzyme Recombinant phage DNA or Bacterial clone Phage clone Phage library Plasmid library

Figure 12.5 Making an intron-lacking gene from eukaryotic mRNA Cell nucleus DNA of eukaryotic gene Exon Intron 1 2 3 4 5 Transcription RNA splicing (removes introns) Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand Breakdown of RNA Synthesis of second DNA strand RNA transcript mRNA Reverse transcriptase cDNA strand cDNA of gene (no introns) Test tube