2. DNA Technology Ch. 20

3. Figure 20.1 An overview of how bacterial plasmids are used to clone genes

4. DNA Technology Recombinant DNA technology –Set of techniques for recombining genes from different sources in vitro and transferring the recombinant DNA to a cell where it is expressed –Typically uses a plasmid as its vector –Same restriction enzymes used to make “sticky end” cuts

5. Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA

6. Action of Restriction Enzymes

7. Restriction Enzymes Cut DNA after specific base sequences = restriction site Protect bacteria –cut up foreign DNA Sticky ends –jagged cut so other DNA can join DNA ligase –makes addition of DNA permanent

8. DNA Technology Biotechnology –Refers to the use of living organisms or components to do practical tasks –Wine industry –Cheese industry –Selective breeding of livestock and crops –Production of antibiotics

9. Cloning a Eukaryotic Gene Isolation of vector & gene-source DNA –Cloning vector is the original plasmid Insertion of DNA into vector –Use of restriction enzymes –May need to make cell competent (E. coli) Introduction of cloning vector into cells –Naked DNA added to culture –Bacteria take in plasmid by transformation

10. Cloning a Eukaryotic Gene Cells reproduce in a culture –Transformed cells are producing new cells with the cloned gene Identification of cell clones –Typically use the R plasmid for ampicillin resistance –Only cells that have transformed can grow on the ampicillin agar –Nucleic acid hybridization

11. Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 1)

12. Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 2)

13. Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)

14. Figure 20.4 Using a nucleic acid probe to identify a cloned gene

15. Chapter 13 Polymerase Chain Reaction Section 1 DNA Technology

16. Chapter 13 Polymerase Chain Reaction Section 1 DNA Technology

17. Polymerase Chain Reaction Use when source of DNA is impure or scarce Clones DNA entirely in vitro Making many copies of a specific segment of DNA (billions of copies in a few hours) Used for DNA analysis –Ancient DNA fragments –DNA from tiny samples –DNA from single embryonic cells –DNA of viral genes

18. Polymerase Chain Reaction Devised in 1985 Starting materials: –DNA polymerase, primers, nucleotides

19. Polymerase Chain Reaction Heat to separate DNA strands –use DNA polymerase from a bacteria that lives in hot springs Cool to allow primers to bind DNA polymerase extends the 3’ end of each primer Multiplies exponentially

20. Southern Blots Hybridization technique that enables researchers to determine the presence of certain nucleotide sequences in a sample of DNA RFLP’s –differences in DNA sequence on homologous chromosomes that result in different patterns of restriction fragment lengths for every species –useful as genetic markers –Inherited following Mendel’s patterns

21. Southern Blots Combination of 5 techniques –Restriction fragment preparation –Electrophoresis –Blotting (DNA bands transferred to nitrocellulose paper) –Hybridization with radioactive probe (attach to gene of interest) –Autoradiography

22. Chapter 13 Gel Electrophoresis Section 1 DNA Technology

23. Chapter 13 DNA Fingerprint Section 1 DNA Technology

24. Figure 20.8 Gel electrophoresis of macromolecules

25. Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

26. Figure 20.10 Restriction fragment analysis by Southern blotting

27. Practical Applications Diagnosis –early detection of disease before symptoms show or even birth –use probes with cloned genes Human gene therapy –traceable genetic disorders –may eventually be correctable –replace defective genes with functional genes –only effective if cells receiving normal allele rapidly reproduce

28. Figure 20.16 One type of gene therapy procedure

29. Practical Applications Environmental –microorganisms to get rid of waste –mining –recycling of wastes and detoxifying –sewage treatment plants

30. Practical Applications Pharmaceutical products –insulin, growth hormone Forensics –blood and tissue type –RFLP and Southern Blots Agricultural –animal husbandry –cellulase

31. Figure 20.17 DNA fingerprints from a murder case

32. Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants

33. Figure 20.x1a Laboratory worker reviewing DNA band pattern

34. Figure 20.x1b DNA study in CDC laboratory