DNA Technology and Genomics

1. DNA Technology and Genomics Chapter 15

2. Learning Objective 1 • How does a typical restriction enzyme cut DNA molecules? • Give examples of the ways in which these enzymes are used in recombinant DNA technology

3. Recombinant DNA Technology • Isolates and amplifies • specific sequences of DNA • incorporates them into vector DNA molecules • Resulting recombinant DNA • is propagated and amplified (cloned) • in organisms such as E. coli

4. Restriction Enzymes • Recognize and cut DNA • at highly specific base sequences • May produce complementary, single-stranded sticky ends

5. Restriction Enzymes

6. Plus HindIII restriction enzyme Sticky ends Fig. 15-1, p. 324

7. KEY CONCEPTS • Recombinant DNA techniques allow scientists to clone many copies of specific genes and gene products

8. Recombinant DNA Vectors • Naturally occurring circular bacteria DNA molecules (plasmids) • Bacterial viruses (bacteriophages)

9. Recombinant DNA Molecules • Construction • ends of DNA fragment and vector • cut with same restriction enzyme • associate by complementary base pairing • DNA ligase • covalently links DNA strands • forms stable recombinant molecule

10. Plasmid from a bacterium DNA of interest from another organism 1 Plasmid and DNA from another organism are cut by the same restriction enzyme (in this example, Hin dIII). This produces molecules with complementary single-stranded ends. Clonable DNA fragment Mix two types of molecules so their sticky ends pair. DNA ligase then forms covalent bonds at junctions, linking fragments. 2 Recombinant DNA 3 Transfer recombinant DNA molecule to host cell, where it is copied and turned on to produce gene product. Fig. 15-2, p. 325

11. Plasmids

12. AatI XbaI Ampicillin resistance Yeast origin of replication HpaI E. coli origin of replication PvuII ClaI Tetracycline resistance URA-3 SalI SmaI BamHI Fig. 15-3a, p. 326

13. Main bacteria DNA Bacterium Plasmid 0.5 μ m Fig. 15-3bc, p. 326

14. Learning Objective 2 • What is the difference between a genomic DNA library, a chromosome library, and a complementary DNA (cDNA) library? • Why would one clone the same eukaryotic gene from both a genomic DNA library and a cDNA library?

15. Libraries (1) • Genomic DNA library • thousands of DNA fragments • all DNA of an organism • Chromosome library • all DNA fragments of a specific chromosome

16. Libraries (2) • Genomic DNA and chromosome libraries • DNA fragments stored in specific bacterial strains • Provide information about genes and encoded proteins

17. Chromosome Library

18. Sites of cleavage Fragment 3 Fragment 1 Fragment 2 Fragment 4 Human DNA Cut with a restriction enzyme 1 2 2 2 2 Produce recombinant DNA Gene for resistance to antibiotic R R R R Transformation 3 Plate with antibiotic- containing medium Bacteria with plasmid live and multiply 4 Bacteria without plasmid fail to grow Fig. 15-4, p. 327

19. Sites of cleavage Fragment 3 Fragment 1 Fragment 2 Fragment 4 Human DNA 1 Cut with a restriction enzyme Produce recombinant DNA 2 2 2 2 Gene for resistance to antibiotic R R R R Transformation 3 Plate with antibiotic- containing medium Bacteria with plasmid live and multiply Bacteria without plasmid fail to grow 4 Stepped Art Fig. 15-4, p. 327

20. cDNA Library • Complementary DNA (cDNA) • produced using reverse transcriptase • makes DNA copies of eukaryotic mRNA • Copies are incorporated into recombinant DNA vectors

21. cDNA

22. Exon Intron Exon Exon Intron DNA in a eukaryotic chromosome Transcription Pre-mRNA RNA processing (remove introns) Mature mRNA Formation of cDNA relies on RNA processing that occurs in the nucleus to yield mature mRNA. Fig. 15-6a, p. 328

23. Reverse transcriptase 1 mRNA cDNA copy of mRNA Degraded RNA 2 cDNA 3 DNA polymerase 4 Double-stranded cDNA Mature mRNA is extracted and purified. Fig. 15-6b, p. 328

24. Introns (1) • Genes regions that do not code for protein • present in eukaryote genomic DNA and chromosome libraries • Genes with introns • can be amplified in bacteria • but protein is not properly expressed

25. Introns (2) • Eukaryotic genes in cDNA libraries • can be expressed in bacteria to produce functional protein products • because introns have been removed from mRNA molecules

26. Learning Objective 3 • What is the purpose of a genetic probe?

27. Genetic Probe • Radioactive DNA or RNA sequence • used to screen recombinant DNA molecules in bacterial cells • to find specific colony with DNA of interest

28. Genetic Probe

29. Bacterial colonies Transfer cells from colonies to nitrocellulose filter 1 Radioactively labeled nucleic acid probe is added Filter with bacteria from colonies; cells are lysed and DNA denatured 2 3 Some radioactive nucleic acid probe molecules become hybridized to DNA of some colonies Exposed X-ray film; dark spots identify colonies with desired DNA 4 Fig. 15-5, p. 328

30. Animation: Use of a Radioactive Probe CLICKTO PLAY

31. Learning Objective 4 • How does the polymerase chain reaction amplify DNA in vitro?

32. Polymerase Chain Reaction (PCR) • Automated in vitro technique • targets a particular DNA sequence by specific primers • clones it using heat-resistant DNA polymerase • Used to analyze tiny DNA samples • from crime scenes, archaeological remains

33. Fig. 15-7, p. 329

34. Learning Objective 5 • What is the difference between DNA, RNA, and protein blotting?

35. Southern Blot • Detects DNA fragments • separates using gel electrophoresis • transfer to nitrocellulose or nylon membrane • Probe is hybridized • by complementary base pairing to DNA bound to membrane • bands of DNA identified by autoradiography or chemical luminescence

36. Gel Electrophoresis

37. Fig. 15-8a, p. 330

38. DNA Cut with restriction enzyme 100 base pairs 200 base pairs 300 base pairs Mixture placed in well Standards of known size + – Origin 300 base pairs Direction of movement 200 base pairs 100 base pairs Gel Fig. 15-8a, p. 330

39. Fig. 15-8b, p. 330

40. Southern Blot

41. Load DNA fragments on gel for electrophoresis. Digest DNA with restriction enzymes. 1 5 2 – + DNA DNA fragments Buffer solution Agarose gel Fig. 15-9, p. 332

42. Buffer solution moves upward, transferring DNA fragments to a DNA-binding filter. 4 DNA fragments are in same location as those on gel. 5 3 Separate DNA by electrophoresis. Longer DNA fragments Weight Nitrocellulose filter Absorbent paper Gel Shorter DNA fragments Wick Buffer 6 7 Fig. 15-9, p. 332

43. Wash filter to remove excess probe and then expose filter to X-ray film; resulting autoradiograph shows hybridized DNA fragments. Place filter and radioactively labeled probe together in sealed bag so it can hybridize. 6 7 Radioactive probe solution Fig. 15-9, p. 332

44. RNA and Proteins • Northern Blot • RNA molecules separated by electrophoresis • transferred to a membrane • Western Blot • Proteins or polypeptides previously separated by gel electrophoresis

45. Learning Objective 6 • What is the chain termination method of DNA sequencing?

46. DNA Sequencing • Yields information about gene structure • and amino acid sequence of encoded proteins • Geneticists compare DNA sequences • with other sequences stored in databases

47. Automated DNA Sequencing • Based on chain termination method • uses dideoxynucleotides • tagged with colored fluorescent dyes • terminates elongation during DNA replication • Gel electrophoresis • separates resulting fragments • laser identifies nucleotide sequence

48. Dideoxynucleotide

49. Dideoxyadenosine triphosphate (ddATP) Fig. 15-10, p. 333

50. Chain Termination Method