Chapter 20 DNA Technology and Genomics

1. Chapter 20DNA Technology and Genomics

2. Chapter 20DNA Technology and Genomics • DNA cloning • DNA probe hybridization • Polymerase Chain Reaction (PCR) • Gel Electrophoresis • Southern Blot • DNA sequencing.

3. Chapter 20DNA Cloning Bacteria have restriction enzymes to attack and destroy invading viral DNA. Restriction enzymes cut DNA at specific nucleotide sequences leaving “sticky ends.” DNA ligase can seal these ends, making recombinant DNA.

4. Chapter 20DNA Cloning Restriction fragments can be put into plasmids. Gene cloning occurs when cells containing these plasmids reproduce. Genes of interest are marked with a radioactive DNA probe.

5. Chapter 20DNA Cloning If a gene is inserted next to a promoter, the bacteria becomes an expression vector. Eukaryotic chromosomes allow for bigger segments of DNA. Eukaryotic cells can also process polypeptides into proteins.

6. Chapter 20DNA Cloning Chopping up the whole genome of an organism produces many DNA fragments containing many genes. Often, the researcher will save all of them, either in bacteria or in viruses. These collections of bacterial clones are called genomic libraries.

7. Chapter 20DNA Probe Hybridization Samples of DNA

8. Chapter 20DNA Probe Hybridization Heat to 95o to denature

9. Chapter 20DNA Probe Hybridization Introduce radioactive probe

10. Chapter 20DNA Probe Hybridization Allow to cool

11. Chapter 20DNA Probe Hybridization √ x x Measure radioactivity

12. Chapter 20Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR) uses DNA polymerase to clone DNA in vitro. In vitro = in a test tube In vivo = in a living organism

13. Chapter 20Gel Electrophoresis DNA Fingerprinting Human DNA contains lots of noncoding sequences that serve no purpose. This “junk DNA” often repeats over and over. No two people (except identical twins) have exactly the same repeats.

14. Chapter 20Gel Electrophoresis DNA Fingerprinting Bill’s chromosome: gene A xxxxx gene B yyy gene C Bob’s chromosome: gene A xx gene B yyyyy gene C

15. Chapter 20Gel Electrophoresis DNA Fingerprinting Restriction enzymes cut DNA at specific places. Bill’s chromosome: gene A xxxxx gene B yyy gene C Bob’s chromosome: gene A xx gene B yyyyy gene C

16. Chapter 20Gel Electrophoresis DNA Fingerprinting Restriction enzymes cut DNA at specific places. Bill’s chromosome: gene A xxxxx gene B yyy gene C Bob’s chromosome: gene A xx gene B yyyyy gene C

17. Chapter 20Gel Electrophoresis DNA Fingerprinting Restriction enzymes cut DNA at specific places. Bill’s chromosome: gene A xxxxx gene B yyy gene C Bob’s chromosome: gene A xx gene B yyyyy gene C

18. Chapter 20Gel Electrophoresis DNA Fingerprinting Restriction enzymes cut DNA at specific places. Bill’s chromosome: Bob’s chromosome:

19. Chapter 20Gel Electrophoresis DNA Fingerprinting Longer fragments travel more slowly through the gel. Bill’s chromosome: Bob’s chromosome:

20. Chapter 20Gel Electrophoresis DNA Fingerprinting Bill: Bob:

21. Chapter 20Gel Electrophoresis DNA Fingerprinting Bill: Bob:

22. Chapter 20Gel Electrophoresis DNA Fingerprinting Bill: Bob:

23. Chapter 20Gel Electrophoresis DNA Fingerprinting Bill: Bob:

24. Chapter 20Gel Electrophoresis

25. Chapter 20Southern Blot The southern blot • Do DNA fingerprinting on an entire genome. • Blot the DNA from the gel to paper with an alkaline solution. This denatures the DNA. • Hybridize with a radioactive probe.

26. Chapter 20Southern Blot The southern blot

27. Chapter 20Southern Blot The southern blot

28. Chapter 20DNA Sequencing • Synthesize DNA 3’ → 5’ • Stop synthesis at random • Dideoxyribonuceotides with dye • Separate fragments by length • Read the colors of the lengths.

29. Chapter 20DNA Sequencing Synthesize DNA 3’ → 5’

30. Chapter 20DNA Sequencing Synthesize DNA 3’ → 5’ 5-AGTACCTG-3

31. Chapter 20DNA Sequencing Synthesize DNA 3’ → 5’ 5-AGTACCTG-3 3-TCATGGAC-5

32. Chapter 20DNA Sequencing Stop the process with didexoyribonucleotides 5-AGTACCTG-3 3-TCATGGAC-5

33. Chapter 20DNA Sequencing Stop the process with didexoyribonucleotides 5-AGTACCTG-3 3-TCATG

34. Chapter 20DNA Sequencing Add a different dye to each didexoyribonucleotide 5-AGTACCTG-3 AT C G

35. Chapter 20DNA Sequencing Stop the synthesis at random times with different dyes 5-AGTACCTG-3 AT C G

36. Chapter 20DNA Sequencing Stop the synthesis at random times with different dyes 5-AGTACCTG-3 3-TCATGG

37. Chapter 20DNA Sequencing Stop the synthesis at random times with different dyes 5-AGTACCTG-3 3-TCATGG 3-T

38. Chapter 20DNA Sequencing Stop the synthesis at random times with different dyes 5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC

39. Chapter 20DNA Sequencing Stop the synthesis at random times with different dyes 5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC 3-TCA

40. Chapter 20DNA Sequencing Stop the synthesis at random times with different dyes 5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC 3-TCA 3-TCATG

41. Chapter 20DNA Sequencing Stop the synthesis at random times with different dyes 5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC 3-TCA 3-TCATG 3-TCA T

42. Chapter 20DNA Sequencing Arrange the fragments by length 5-AGTACCTG-3 3-TCATGG 3-T 3-TCATGGAC 3-TCA 3-TCATG 3-TCA T

43. Chapter 20DNA Sequencing Arrange the fragments by length 3-T 3-TC 3-TCA 3-TCA T 3-TCATG 3-TCATGG 3-TCATGGA 3-TCATGGAC

44. Chapter 20DNA Sequencing Read the colors 3-T 3-TC 3-TCA 3-TCA T 3-TCATG 3-TCATGG 3-TCATGGA 3-TCATGGAC

45. Chapter 20DNA Sequencing

46. Chapter 20DNA Sequencing

47. Chapter 20DNA Technology and Genomics RFLPs (“RIF-lips”), or restriction fragment length polymorphisms, are differences in homologous chromosomes that give different length restriction fragments. Chromosome walking means finding where fragments of DNA overlapped in the genome.

48. Chapter 20DNA Technology and Genomics Genomics is the systematic study of entire genomes. Proteomics is the study of all the proteins encoded by a genome. Single nucleotide polymorphisms (SNPs) are useful markers for studying variation.

49. Chapter 20DNA Technology and Genomics Uses of DNA Technology: Testing for genetic diseases Large scale production of drugs Gene therapy Forensics Genetic engineering. .