1. Molecular Genetic Analysis and Biotechnology
Benjamin A. Pierce
GENETICS
A Conceptual Approach
FIFTH EDITION
CHAPTER 19
Molecular Genetic Analysis and Biotechnology
© 2014 W. H. Freeman and Company

2. Blindness affects 45 million people throughout the world
Blindness affects 45 million people throughout the world. Genetic engineering is now being used to treat patients with Leber congenital amaurosis, a genetic form of blindness. [Paul Doyle/Alamy Images.]

3. Techniques of Molecular Genetics Have Revolutionized Biology
Recombinant DNA Technology (Genetic Engineering)
Techniques for locating, isolating, altering, and studying DNA segments
The Molecular Genetics Revolution
Biotechnology: the use of these techniques to develop new products
Working at the Molecular Level

4. Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
First step: isolate DNA segment or gene from remaining DNA
Cutting and joining DNA fragments—restriction enzymes
Viewing DNA fragments
Locating DNA fragments with southern blotting and probes

5. Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
First step: isolate DNA segment or gene from remaining DNA
Cutting and joining DNA fragments—restriction enzymes
Viewing DNA fragments
Locating DNA fragments with southern blotting and probes

6. Cutting and Joining DNA Fragments
Restriction enzymes: recognizing and cutting DNA at specific nucleotide sequences
Type II restriction enzyme: most useful enzyme
By adding methyl groups to the recognition sequence to protect itself from being digested by its own enzyme in bacteria

7. Cutting and Joining DNA Fragments
Cohesive ends: fragments with short, single-stranded overhanging ends
Blunt ends: even-length ends from both single strands

9. Figure 19.2a Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends.

10. Figure 19.2b Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends.

11. Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
First step: isolate DNA segment or gene from remaining DNA
Cutting and joining DNA fragments—restriction enzymes
Viewing DNA Fragments
Locating DNA fragments with southern blotting and probes

12. Viewing DNA Fragments
Gel electrophoresis
Autoradiography

13. Figure Gel electrophoresis can be used to separate DNA molecules on the basis of their size and electrical charge. [Photograph courtesy of Carol Eng.]

14. Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
First step: isolate DNA segment or gene from remaining DNA
Cutting and joining DNA fragments—restriction enzymes
Viewing DNA fragments
Locating DNA fragments with southern blotting and probes

15. Locating DNA Fragments with Southern Blotting and Probes
Probe: DNA or RNA with a base sequence complementary to a sequence in the gene of interest

16. Figure 19.4 Southern blotting and hybridization with probes can locate a few specific fragments in a large pool of DNA.

17. Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
Cloning genes
Application: the genetic engineering of plants with pesticides
Amplifying DNA fragments with the polymerase chain reaction

18. Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
Cloning genes
Application: the genetic engineering of plants with pesticides
Amplifying DNA fragments with the polymerase chain reaction

19. Cloning Genes
Gene cloning: amplifying a specific piece of DNA via a bacteria cell
Cloning vector: a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell

20. Figure 19.5 An idealized cloning vector has an origin of replication, one or more selectable markers, and one or more unique restriction sites.

21. Cloning Genes Plasmid vectors
Plasmids: circular DNA molecules from bacteria
Insert foreign DNA into plasmid using restriction enzymes
Linkers: synthetic DNA fragments containing restriction sites
Transformation of host cells with plasmids
Screening cells for recombinant plasmids
Selectable markers are used to confirm whether the cells have been transformed or not

22. Figure 19. 6 The pUC19 plasmid is a typical cloning vector
Figure 19.6 The pUC19 plasmid is a typical cloning vector. It contains a cluster of unique restriction sites, an origin of replication, and two selectable markers—an ampicillin-resistance gene and a lacZ gene.

23. Figure 19.7 A foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes.

24. Figure 19.8 The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin-resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

25. Cloning Genes Other gene vectors: Cosmids
Bacterial Artificial Chromosomes (BACs)
Yeast Artificial Chromosome
Ti plasmid

27. Figure To ensure transcription and translation, a foreign gene may be inserted into an expression vector—in this example, an E. coli expression vector.

28. Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
Cloning genes
Application: the genetic engineering of plants with pesticides
Amplifying DNA fragments with the polymerase chain reaction

29. Application: The Genetic Engineering of Plants with Pesticide
Bacillus thuringienis
Bt gene
Agrobacterium tumefaciens

31. Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
Cloning genes
Application: the genetic engineering of plants with pesticides
Amplifying DNA fragments with the polymerase chain reaction

32. Amplifying DNA Fragments with the Polymerase Chain Reaction (PCR)
The PCR reaction
Taq polymerase: stable DNA polymerase at high temperature
Reverse-transcription PCR

33. Figure 19.11 The polymerase chain reaction is used to amplify even very small samples of DNA.

34. Figure 19.11 The polymerase chain reaction is used to amplify even very small samples of DNA.

35. Amplifying DNA Fragments with the Polymerase Chain Reaction (PCR)
Limitations of PCR
Prior knowledge of target DNA
Contamination
Accuracy
Amplified fragments are less than 2 kb

36. Amplifying DNA fragments with the polymerase chain reaction (PCR)
Applications of PCR
Real-time PCR: quantitatively determining the amount of DNA amplified as the reaction proceeds

37. Molecular Techniques Can Be Used to Find Genes of Interest
Gene libraries
In situ hybridization
Positional cloning
Application: isolating the gene for cystic fibrosis

38. Molecular Techniques Can Be Used to Find Genes of Interest
Gene libraries
In situ hybridization
Positional cloning
Application: isolating the gene for cystic fibrosis

39. Gene Libraries
DNA library: a collection of clones containing all the DNA fragments from one source
Creating a genomic DNA library
cDNA libraries: consisting only of those DNA sequences that are transcribed into mRNA

40. Figure 19.14 A genomic library contains all of the DNA sequences found in an organism’s genome.

41. Gene Libraries Screening DNA libraries Plating clones of the library
Probing plated colonies or plaques

43. Molecular Techniques Can Be Used to Find Genes of Interest
Gene libraries
In situ hybridization
Positional cloning
Application: isolating the gene for cystic fibrosis

44. In Situ Hybridization
DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell
FISH

46. Molecular Techniques Can Be Used to Find Genes of Interest
Gene libraries
In situ hybridization
Positional cloning
Application: isolating the gene for cystic fibrosis

47. Positional Cloning
Isolating genes on the basis of their position on a genetic map
Chromosome walking
Chromosome jumping

49. Molecular Techniques Can Be Used to Find Genes of Interest
Gene libraries
In situ hybridization
Positional cloning
Application: isolating the gene for cystic fibrosis

50. Application: Isolating the Gene for Cystic Fibrosis
Autosomal recessive disorder
Characterized by chronic lung infections, insufficient pancreatic enzyme production, and increased salt concentration in sweat

51. Figure 19.20 The gene for cystic fibrosis was located by positional cloning.

52. Figure A candidate for the cystic fibrosis gene is expressed in pancreatic, respiratory, and sweat-gland tissues—tissues that are affected by the disease. Shown is a Northern blot of mRNA produced by the candidate gene in different tissues. These data provided evidence that the candidate gene is in fact the gene that causes cystic fibrosis. [After J. R. Riordan et al., Science 245:1066–1073, 1989.]

53. DNA Sequences Can Be Determined and Analyzed
Restriction Fragment Length Polymorphisms (RFLPs)
DNA sequencing
Next-generation sequencing technologies
DNA fingerprinting
Application: identifying people who died in the collapse of the World Trade Center

54. DNA Sequences Can Be Determined and Analyzed
Restriction Fragment Length Polymorphisms (RFLPs)
DNA sequencing
Next-generation sequencing technologies
DNA fingerprinting
Application: identifying people who died in the collapse of the World Trade Center

55. Restriction Fragment Length Polymorphisms
Some DNA fragments have different restriction sites due to mutation for the same restriction enzyme
Causes polymorphisms within a population

56. Figure Restriction fragment length polymorphisms are genetic markers that can be used in mapping.

57. Figure Restriction fragment length polymorphisms can be used to detect linkage. There is a close correspondence between the inheritance of the RFLP alleles and the presence of Huntington disease, indicating that the genes that encode the RFLP and Huntington disease are closely linked.

58. DNA Sequences Can Be Determined and Analyzed
Restriction Fragment Length Polymorphisms (RFLPs)
DNA sequencing
Next-generation sequencing technologies
DNA fingerprinting
Application: identifying people who died in the collapse of the World Trade Center

59. DNA Sequencing Sanger’s dideoxy-sequencing method
Dideoxyribonucleoside triphosphate (ddNTP) lacks a 3′-oh group, which terminates DNA synthesis

60. Figure The dideoxy sequencing reaction requires a special substrate for DNA synthesis. (a) Structure of deoxyribonucleoside triphosphate, the normal substrate for DNA synthesis. (b) Structure of dideoxyribonucleoside triphosphate, which lacks an OH group on the 3′-carbon atom.

61. Figure 19.25 The dideoxy method of DNA sequencing is based on the termination of DNA synthesis.

63. DNA Sequences Can Be Determined and Analyzed
Restriction Fragment Length Polymorphisms (RFLPs)
DNA sequencing
Next-generation sequencing technologies
DNA fingerprinting
Application: identifying people who died in the collapse of the World Trade Center

64. Next-Generation Sequencing Technologies
Pyrosequencing
Illumina sequencing
Third-generation sequencing

66. DNA Sequences Can Be Determined and Analyzed
Restriction Fragment Length Polymorphisms (RFLPs)
DNA sequencing
Next-generation sequencing technologies
DNA fingerprinting
Application: identifying people who died in the collapse of the World Trade Center

67. DNA Fingerprinting (DNA Profiling)
Microsatellites: (short tandem repeats, STRs) variable number of copies of repeat sequences possessed by many organisms
Detected by PCR
Fragments represented as peaks on a graph
Homozygotes: single tall peak
Heterozygotes: two shorter peaks

71. DNA Sequences Can Be Determined and Analyzed
Restriction Fragment Length Polymorphisms (RFLPs)
DNA sequencing
Next-generation sequencing technologies
DNA fingerprinting
Application: identifying people who died in the collapse of the World Trade Center

72. Application: Identifying People Who Died in the Collapse of the World Trade Center
Usual means of victim ID were of little use with WTC remains
Used DNA fingerprinting
Also carried out on mitochondrial DNA
INSERT FIG 19.31

73. Molecular Techniques Are Increasingly Used to Analyze Gene Function
Forward and reverse genetics
Creating random mutations
Site-directed mutagenesis
Transgenic animals
Knockout mice
Silencing genes with RNAi
Application: Using RNAi for the treatment of human disease

74. Forward and Reverse Genetics
Forward genetics: Begins with a phenotype to a gene that encodes the phenotype
Reverse genetics: Begins with a gene of unknown function, first inducing mutations and then checking the effect of the mutation on the phenotype

75. Creating Random Mutations
A means to increase the number of mutants in an experimental population
Use mutagenic agents: radiation, chemical mutagens, transposable elements

76. Site-Directed Mutagenesis
Oligonucleotide-directed mutagenesis

77. Figure Oligonucleotide-directed mutagenesis is used to study gene function when appropriate restriction sites are not available.

78. Transgenic Animals
An organism permanently altered by the addition of a DNA sequence to its genome
Transgene

80. Knockout Mice A normal gene of the mouse has been fully disabled
Knock-in mice: a mouse carries an inserted DNA sequence at specific locations

82. Silencing Genes with RNAi
siRNAs
Process called RNA interference (RNAi)

84. Application: Using RNAi for the Treatment of Human Disease
High cholesterol
RNAi could be used to reduce the levels of ApoB and blood cholesterol in nonhuman primates

85. Biotechnology Harnesses the Power of Molecular Genetics
Pharmaceutical products
Specialized bacteria
Agriculture products
Genetic testing
Gene therapy