Bacterium

1. Bacterium Cell containing geneof interest Gene inserted into plasmid 4 2 3 1 Gene of interest Plasmid Bacterialchromosome DNA ofchromosome RecombinantDNA (plasmid) Plasmid put into bacterial cell Recombinatebacterium Host cell grown in culture,to form a clone of cellscontaining the “cloned”gene of interest Gene of interest Protein expressedby gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on protein Basic research on gene Gene used to alterbacteria for cleaningup toxic waste Gene for pestresistance inserted into plants Human growth hormone treatsstunted growth Protein dissolvesblood clots in heartattack therapy Figure 20.2 Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes

2. 3 1 2 Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA Restriction site 5 3 G A A T T C DNA 5 3 C T T A A G Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow G A A T T C C T T A A G Sticky end A A T T C G G DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. C T T A A Fragment from differentDNA molecule cut by thesame restriction enzyme G A A T T C A A T T C G C T T A A T T A A C G G One possible combination DNA ligaseseals the strands. Recombinant DNA molecule

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

4. Genome cut up withrestriction enzyme Recombinantplasmid Recombinantphage DNA or Bacterialclone Phageclone Plasmid library Phage library Genomic libraries

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

6. Table 12.6 Some protein products of recombinant DNA technology

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

8. 1 2 3 3 5 Target sequence 3 5 Genomic DNA 3 5 Denaturation: Heat briefly to separate DNA strands 5 3 Annealing: Cool to allow primers to hydrogen-bond. Cycle 1 yields 2 molecules Primers Extension: DNA polymerase adds nucleotidesto the 3 end of each primer Newnucleo-tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence PCR

9. PCR

10. Primitive PCR machine

12. Applications for PCR • DNA cloning for sequencing • Functional analysis of genes • Diagnosis of genetic diseases • ID genetic fingerprints (i.e. forensics and paternity testing) • Detection and diagnosis of infectious diseases (e.g. H1N1)

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

14. Lane 1 – Father Lane 2 – Child Lane 3 – Mother The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new and unique fingerprint.

15. Gel box and power source

16. Gel Electrophoresis Plate

17. Blood from defendant’s clothes Victim’s blood (V) Defendant’s blood (D) 4 g 8 g V Jeans D shirt DNA fingerprints from a murder case

18. “Pharm” animals