1. Molecular Cell Biology Chapter 5. Molecular Genetic Techniques
Professor Dawei Li
Textbook: MOLECULAR CELL BIOLOGY 6th Ed
Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira
Part 2. Genetics and Molecular Biology
Chapter 5. Molecular Genetic Techniques

3. © 2008 W. H. Freeman and Company
CHAPTER 5
Molecular Genetic Techniques
© 2008 W. H. Freeman and Company

4. Teaching Plan- Chapter 5 April 16, 2015
Activity
Scheduled Action/ timing
Student Presentation
7 minutes
Lecture by Prof Li. Chapter 5
As per content requirement
Lecture Continued
Video-- Expression Cloning of Receptors
Plasmid Cloning(Section 5.2)
5 minutes
4 minutes
Assignment- Review chapter/section covered in class,
Quiz in next class, 5-10 minutes
Each group will present a either a topic from Chapter or a latest research paper as per chapter title. One group in each class.
Group 1 presentation in immediate next class. Time limit 10 minutes.

5. 5.1 Genetic Analysis of Mutations to Identify and Study Genes
OUTLINE
5.1 Genetic Analysis of Mutations to Identify and Study Genes
5.2 DNA cloning and Characterization
5.3 Using Cloned DNA Fragments to Study Gene Expression
5.4 Identifying and Locating Human Disease Genes
5.5 Inactivating the Function of Specific Genes in Eukaryotes

6. DNA Cloning and Characterization 176- 190
SECTION 5.2
DNA Cloning and Characterization

7. Replication Vector +DNA Fragment
P. No DNA Cloning and Characterization
Replication Vector +DNA Fragment
Recombinant DNA
Replication in Host Cells
Characterization and Manipulation of Purified DNA

8. Cutting DNA Molecules into Small Fragments
P.No.176 FIGURE 5-11
Cleavage of DNA by the restriction enzyme EcoRI

10. Inserting DNA Fragments into Vectors
178 FIGURE 5-12
Ligation of restriction fragments with complementary sticky ends

11. 178 FIGURE 5-13
Basic components of a plasmid cloning vector that can replicate within an E.coli cell
E.Coli Plasmid Vectors Are Suitable for Cloning Isolated DNA Fragment

12. 179 FIGURE 5-14
DNA cloning in a plasmid vector permits amplification of a DNA fragment

13. Video- Expression Cloning of Receptors

14. FIGURE 5-14(a)
DNA cloning in a plasmid vector permits amplification of a DNA fragment

15. FIGURE 5-14(b)
DNA cloning in a plasmid vector permits amplification of a DNA fragment

16. Video – Plasmid Cloning

17. cDNAs Prepared by Reverse Transcription of Cellular mRNAs Can Be Cloned to Generate cDNA Libraries
180 FIGURE 5-15
A cDNA library contains representative copies of cellular mRNA sequences

18. FIGURE 5-15(a)
A cDNA library contains representative copies of cellular mRNA sequences

19. FIGURE 5-15(b)
A cDNA library contains representative copies of cellular mRNA sequences

20. FIGURE 5-15(c)
A cDNA library contains representative copies of cellular mRNA sequences

21. FIGURE 5-15(d)
A cDNA library contains representative copies of cellular mRNA sequences

22. FIGURE 5-15(e)
A cDNA library contains representative copies of cellular mRNA sequences

23. DNA Libraries Can Be Screened by Hybridization to an Oligonucleotide Probe
182 FIGURE 5-16
cDNA libraries can be screened with a radiolabeled probe to identify a clone of interest

24. Yeast Genomic Libraries Can Be Constructed with Shuttle Vectors and Screened by Functional Complementation
183 FIGURE 5-17
A yeast genomic library can be constructed in a plasmid shuttle vector that can replication in yeast and E.coli

25. FIGURE 5-17(a)
A yeast genomic library can be constructed in a plasmid shuttle vector that can replication in yeast and E.coli

26. FIGURE 5-17(b)
A yeast genomic library can be constructed in a plasmid shuttle vector that can replication in yeast and E.coli

27. 184 FIGURE 5-18
Screening of a yeast genomic library by functional complementation can identify clones carrying the normal form of a mutant yeast gene

28. Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments
185 FIGURE 5-19
Gel electrophoresis separates DNA molecules of different lengths

29. FIGURE 5-19(d)
Gel electrophoresis separates DNA molecules of different lengths

30. Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method
FIGURE 5-20
Structures of deoxyribonucleoside triphosphate (dNTP) and dideoxyribonucleoside triphosphate (ddNTP)

31. EXPERIMENTAL FIGURE 5-21(a)
Cloned DNAs can be sequenced by the Sanger method, using fluorescent-tagged dideoxyribonucleoside triphosphates (ddNTPs)

32. EXPERIMENTAL FIGURE 5-21(b)
Cloned DNAs can be sequenced by the Sanger method, using fluorescent-tagged dideoxyribonucleoside triphosphates (ddNTPs)

33. EXPERIMENTAL FIGURE 5-21(c)
Cloned DNAs can be sequenced by the Sanger method, using fluorescent-tagged dideoxyribonucleoside triphosphates (ddNTPs)

34. P.No 187 Strategies for Assembling Whole Genome Sequences
FIGURE 5-22
Two Strategies for Assembling Whole Genome Sequences

35. Dideoxy Sequencing of DNA
Video
Dideoxy Sequencing of DNA

36. The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture
EXPERIMENTAL FIGURE 5-23
The polymerase chain reaction (PCR) is widely used to amplify DNA regions of known sequence

37. Polymerase Chain Reaction
Video
Polymerase Chain Reaction

38. P.No 189 Direct Isolation of a Specific Segment of Genomic DNA
EXPERIMENTAL FIGURE 5-24
A specific target region in total genomic DNA can be amplified by PCR for use in cloning

39. Tagging of Genes by Insertion Mutations
189 EXPERIMENTAL FIGURE 5-25
The genomic sequence at the insertion site of a transposon is revealed by PCR amplification and sequencing

40. Review KEY CONCEPT OF SECTION 5.2 ( Assignment)
DNA Cloning and Characterizaiton(p190)

41. Teaching Plan- Chapter 5 April 23 , 2015
Activity
Scheduled Action/ timing
Student Presentation Group 1 (April 29, 2015) tion Discussion
Student Presentation
Each group will present a either a topic from Chapter or a latest research paper as per chapter title. One group in each class.
Lecture by Prof Li. Chapter 5
As per content requirement
Lecture Continued
Video– Synthesizing an oligonucleotide Array
Screening for Patterns for Gene Therapy
5 minutes
4 minutes
Assignment- Review section covered in class,
Student Presentation Group 1 (April 29, 2015)

42. P.No 191 5.3 Use Cloned DNA Fragments to Study Gene Expression
EXPERIMENTAL FIGURE 5-26
Southern blot technique can detect a specific DNA fragment in a complex mixture of restriction fragments

43. Hybridization Techniques Permit Detection of Specific DNA Fragments and mRNAs
192 EXPERIMENTAL FIGURE 5-27
Northern blot analysis reveals increased expression of β-globin mRNA in differentiated erthroleukemia cells

44. In Situ Hybridization
193 EXPERIMENTAL FIGURE 5-28
In situ hybridization can detect activity of specific genes in whole and sectioned embryos

45. Page No. 194 Video Synthesizing an oligonucleotide Array
Screening for Patterns for Gene Therapy

46. P.No 194 Using Microarrays to Compare Gene Expression under Different Conditions
EXPERIMENTAL FIGURE 5-29(a)
DNA microarray analysis can reveal differences in gene expression in fibroblasts under different experimental conditions

47. 194 EXPERIMENTAL FIGURE 5-29(b)
DNA microarray analysis can reveal differences in gene expression in fibroblasts under different experimental conditions

48. 195 Cluster Analysis of Multiple Expression Experiments Identifies Co-regulated Genes
EXPERIMENTAL FIGURE 5-30
Cluster analysis of data from multiple microarray expression experiments can identify co-regulated genes

49. E.Coli Expression Systems Can Produce Large Quantities of Proteins from Cloned Genes
P.No 195 EXPERIMENTAL FIGURE 5-31
Some eukaryotic proteins can be produced in E.coli cells from plasmid vectors containing the lac promoter

50. P.No 195 EXPERIMENTAL FIGURE 5-31(a)
Some eukaryotic proteins can be produced in E.coli cells from plasmid vectors containing the lac promoter

51. 196 EXPERIMENTAL FIGURE 5-31(b)
Some eukaryotic proteins can be produced in E.coli cells from plasmid vectors containing the lac promoter

52. Teaching Plan- Chapter 5 April 30 , 2015
Activity
Scheduled Action/ timing
Group 1- Students presentations
7 minutes
Lecture by Prof Li. Chapter 5
As per content requirement
Lecture Continued
Video– Synthesizing an oligonucleotide Array
Screening for Patterns for Gene Therapy
5 minutes
4 minutes
Student Presentation Group 2 (May 5 , 2015)

53. 196 Plasmid Expression Vectors Can Be Designed for Use in Animal Cells
EXPERIMENTAL FIGURE 5-32(a)
Transient and stable transfection with specially designed plasmid vectors permit expression of cloned genes in cultured animal cells

54. Page 196 EXPERIMENTAL FIGURE 5-32(b)
Transient and stable transfection with specially designed plasmid vectors permit expression of cloned genes in cultured animal cells

55. P.No 197 Retroviral Expression Systems
EXPERIMENTAL FIGURE 5-33
Retroviral vectors can be used for efficient integration of cloned genes into the mammalian genome

56. 198 Gene and Protein Tagging
EXPERIMENTAL FIGURE 5-34
Gene and protein tagging facilitate cellular localization of proteins expressed from cloned genes

57. Review KEY CONCEPTS OF SECTION 5.3
Using Cloned DNA Fragments to Study Gene Expression(p198)

58. Page 199 5.4 Identifying and Locating Human Disease Genes

59. Page 200 Many Inherited Diseases Show One of Three Major Patterns of Inheritance
FIGURE 5-35
Three common inheritance patterns of human genetic diseases

60. Teaching Plan- Chapter 5 May 5 , 2015
Activity
Scheduled Action/ timing
Group 2- Students presentations
7 minutes
Lecture by Prof Li. Chapter 5
As per content requirement
Lecture Continued
Video– Microinjection of ES cells into a blastocyst
Screening for Patterns for Gene Therapy
3 minutes
4 minutes
Student Presentation on 7th May:
Chapter 25
Topic : Immunotherapy of cancer
Related topic:
CART- Chimeric Antigen Receptor T Cells
Presenters:
Sehar, Lorna, Janie, Harry, Yeasin
Preview Chapter 6

61. Content of Presentation
1. Chapter Name – (You have chosen the topic/ paper from)
2. Introduction
Background
Significance- Present status
Future Prospects

62. Page 201 DNA Polymorphisms Are Used in Linkage-Mapping Human Mutations
Restriction fragment length polymorphisms
EXPERIMENTAL FIGURE 5-36(a)
Restriction fragment length polymorphisms (RFLPs) can be followed like genetic markers

63. 201 EXPERIMENTAL FIGURE 5-36(b)
Restriction fragment length polymorphisms (RFLPs) can be followed like genetic markers

64. Page 202 Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan
FIGURE 5-37
Linkage disequilibrium studies of human populations can be used to map genes at high resolution

65. Page 203 Further Analysis Is Needed to Locate a Disease Gene in Cloned DNA
FIGURE 5-38
The relationship between the genetic and physical maps of a human chromosome

66. Review KEY CONCEPTS OF SECTION 5.4
Identifying and Locating Human Disease Genes(p204)

67. 205 5.5 Inactivating the Function of Specific Genes in Eukaryotes
Gene Knockout
Normal Yeast Genes Can Be Replaced with Mutant Alleles by Homologous Recombination
EXPERIMENTAL FIGURE 5-39(a)
Homologous recomnination with fransfected disruption constructs can inactivate specific target genes in yeast

68. Study essential genes by conditional knockout
Gal1 Promoter-Essential Gene
Grow in Galactose medium
Grow in Glucose medium
Mutant phenotype
205 EXPERIMENTAL FIGURE 5-39(b)
Homologous recomnination with fransfected disruption constructs can inactivate specific target genes in yeast

69. 206 Specific Genes Can Be Permanently Inactivated in the Germ Line of Mice
EXPERIMENTAL FIGURE 5-40(a)
Isolation of mouse ES cells with a gene-targeted disruption is the first stage in production of knockout mice

70. 206 EXPERIMENTAL FIGURE 5-40(b)
Isolation of mouse ES cells with a gene-targeted disruption is the first stage in production of knockout mice

71. Microinjection of ES cells into a blastocyst
Video
Microinjection of ES cells into a blastocyst

72. 207 EXPERIMENTAL FIGURE 5-41
ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice

73. EXPERIMENTAL FIGURE 5-41(a)
ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice

74. EXPERIMENTAL FIGURE 5-41(b)
ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice

75. EXPERIMENTAL FIGURE 5-41(c)
ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice

76. Conditional Knockout: To Study Embryonic Lethal Essential Gene KO

77. Somatic Cell Recombination Can Inactivate Genes in Specific Tissues
208 EXPERIMENTAL FIGURE 5-42
The loxP-Cre recombination system can knock out genes in specific cell types

78. Creating a Transgenic Mouse
Video
Creating a Transgenic Mouse

79. Dominant-Negative Alleles Can Functionally Inhibit Some Genes
209 EXPERIMENTAL FIGURE 5-43
Transgenic mice are produced by random integration of a foreign gene into the mouse germ

80. 210 FIGURE 5-44
Inactivation of the function of a wild-type GTPase by the action of a dominant-negative mutant allele

81. RNA Interference Causes Gene Inactivation by Destroying the Corresponding mRNA
211 EXPERIMENTAL FIGURE 5-45
RNA interference (RNAi) can functionally inactivate genes in C.elegans and other organisms

82. 211 EXPERIMENTAL FIGURE 5-45(a)
RNA interference (RNAi) can functionally inactivate genes in C.elegans and other organisms

83. EXPERIMENTAL FIGURE 5-45(b)
RNA interference (RNAi) can functionally inactivate genes in C.elegans and other organisms

84. EXPERIMENTAL FIGURE 5-45(c)
RNA interference (RNAi) can functionally inactivate genes in C.elegans and other organisms

85. Review KEY CONCEPTS OF SECTION 5.5
Inactivating the Function of Specific Genes in Eukaryotes(p211)

86. Discussion:
Answer Chapter 5 Questions
Homework: Review Chapter 5
Key Terms (p212)
Concepts p212 (will be tested in Final)
Analyzing the data p
(These will be tested in Final)
Next Thursday Chapter 5 Test.

87. KEY WORDS Allete 等位基因 Mutation 突变 Mutagen 诱变剂 Genotype 基因型 Wild type
野生型
Phenotype 表型
Haploid
单倍体
Diploid
二倍体
Heterozygous
杂合子
Homozygous
纯合子
Recessive 隐性
Dominant 显性
Point mutation
点突变
Gamete 配子
Mitosis
有丝分裂