Molecular Cell BiologyProfessor Dawei Li daweili@sjtu.edu.cn 3420-4744

Part 2. Genetics and Molecular Biology

Textbook: MOLECULAR CELL BIOLOGY 6th EdLodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira

Chapter 5. Molecular Genetic Techniques

CHAPTER 5Molecular Genetic Techniques

CHAPTER 5Molecular Genetic Techniques

© 2008 W. H. Freeman and Company

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 minutes4 minutes

Assignment- Review chapter/section covered in class,

Quiz in next class, 5-10 minutes

Student PresentationEach group will present a either a topic from Chapter 16- 25 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.

Teaching Plan- Chapter 5 April 16, 2015

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

SECTION 5.2

DNA Cloning and Characterization 176- 190

P. No 176 5.2 DNA Cloning and Characterization

Replication Vector +DNA Fragment

Recombinant DNA

Replication in Host Cells

Characterization and Manipulation of Purified DNA

P.No.176 FIGURE 5-11Cleavage of DNA by the restriction enzyme EcoRI

Cutting DNA Molecules into Small Fragments

178 FIGURE 5-12Ligation of restriction fragments with complementary sticky ends

Inserting DNA Fragments into Vectors

178 FIGURE 5-13Basic 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

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

Video- Expression Cloning of Receptors

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

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

Video – Plasmid Cloning

180 FIGURE 5-15A cDNA library contains representative copies of cellular mRNA sequences

cDNAs Prepared by Reverse Transcription of Cellular mRNAs Can Be Cloned to Generate cDNA Libraries

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

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

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

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

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

182 FIGURE 5-16cDNA libraries can be screened with a radiolabeled probe to identify a clone of interest

DNA Libraries Can Be Screened by Hybridization to an Oligonucleotide Probe

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

Yeast Genomic Libraries Can Be Constructed with Shuttle Vectors and Screened by Functional Complementation

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

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

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

185 FIGURE 5-19Gel electrophoresis separates DNA molecules of different lengths

Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments

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

FIGURE 5-20Structures of deoxyribonucleoside triphosphate (dNTP) and dideoxyribonucleoside triphosphate (ddNTP)

Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method

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

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

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

FIGURE 5-22Two Strategies for Assembling Whole Genome Sequences

P.No 187 Strategies for Assembling Whole Genome Sequences

Video

Dideoxy Sequencing of DNA

EXPERIMENTAL FIGURE 5-23The polymerase chain reaction (PCR) is widely used to amplify DNA regions of known sequence

The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture

Video

Polymerase Chain Reaction

EXPERIMENTAL FIGURE 5-24A specific target region in total genomic DNA can be amplified by PCR for use in cloning

P.No 189 Direct Isolation of a Specific Segment of Genomic DNA

189 EXPERIMENTAL FIGURE 5-25The genomic sequence at the insertion site of a transposon is revealed by PCR amplification and sequencing

Tagging of Genes by Insertion Mutations

Review KEY CONCEPT OF SECTION 5.2 ( Assignment)

DNA Cloning and Characterizaiton(p190)

Activity Scheduled Action/ timing

Student Presentation Group 1 (April 29, 2015) tion Discussion

Student PresentationEach group will present a either a topic from Chapter 16- 25 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

1. Video– Synthesizing an oligonucleotide Array

2. Screening for Patterns for Gene Therapy

5 minutes4 minutes

Assignment- Review section covered in class,

Student Presentation Group 1 (April 29, 2015)

Teaching Plan- Chapter 5 April 23 , 2015

P.No 191 5.3 Use Cloned DNA Fragments to Study Gene Expression

EXPERIMENTAL FIGURE 5-26Southern blot technique can detect a specific DNA fragment in a complex mixture of restriction fragments

192 EXPERIMENTAL FIGURE 5-27Northern blot analysis reveals increased expression of β-globin mRNA in differentiated erthroleukemia cells

Hybridization Techniques Permit Detection of Specific DNA Fragments and mRNAs

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

In Situ Hybridization

Page No.194 Video

1. Synthesizing an oligonucleotide Array

2. Screening for Patterns for Gene Therapy

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

P.No 194 Using Microarrays to Compare Gene Expression under Different Conditions

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

EXPERIMENTAL FIGURE 5-30Cluster analysis of data from multiple microarray expression experiments can identify co-regulated genes

195 Cluster Analysis of Multiple Expression Experiments Identifies Co-regulated Genes

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

E.Coli Expression Systems Can Produce Large Quantities of Proteins from Cloned Genes

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

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

Activity Scheduled Action/ timingGroup 1- Students presentations 7 minutes

Lecture by Prof Li. Chapter 5 As per content requirement Lecture Continued 1. Video– Synthesizing an

oligonucleotide Array 2. Screening for Patterns for Gene

Therapy

5 minutes4 minutes

Student Presentation Group 2 (May 5 , 2015)

Teaching Plan- Chapter 5 April 30 , 2015

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

196 Plasmid Expression Vectors Can Be Designed for Use in Animal Cells

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

EXPERIMENTAL FIGURE 5-33Retroviral vectors can be used for efficient integration of cloned genes into the mammalian genome

P.No 197 Retroviral Expression Systems

EXPERIMENTAL FIGURE 5-34Gene and protein tagging facilitate cellular localization of proteins expressed from cloned genes

198 Gene and Protein Tagging

Review KEY CONCEPTS OF SECTION 5.3

Using Cloned DNA Fragments to Study Gene Expression(p198)

Page 199 5.4 Identifying and Locating Human Disease Genes

FIGURE 5-35Three common inheritance patterns of human genetic diseases

Page 200 Many Inherited Diseases Show One of Three Major Patterns of Inheritance

Activity Scheduled Action/ timingGroup 2- Students presentations 7 minutes

Lecture by Prof Li. Chapter 5 As per content requirement Lecture Continued 1. Video– Microinjection of ES cells

into a blastocyst2. Screening for Patterns for Gene

Therapy

3 minutes4 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

Teaching Plan- Chapter 5 May 5 , 2015

Content of Presentation

• 1. Chapter Name – (You have chosen the topic/ paper from)

• 2. Introduction• Background • Significance- Present status• Future Prospects

EXPERIMENTAL FIGURE 5-36(a)Restriction fragment length polymorphisms (RFLPs) can be followed like genetic markers

Page 201 DNA Polymorphisms Are Used in Linkage-Mapping Human Mutations

Restriction fragment length polymorphisms

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

FIGURE 5-37Linkage disequilibrium studies of human populations can be used to map genes at high resolution

Page 202 Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan

FIGURE 5-38The relationship between the genetic and physical maps of a human chromosome

Page 203 Further Analysis Is Needed to Locate a Disease Gene in Cloned DNA

Review KEY CONCEPTS OF SECTION 5.4

Identifying and Locating Human Disease Genes(p204)

205 5.5 Inactivating the Function of Specific Genes in Eukaryotes

Gene Knockout

EXPERIMENTAL FIGURE 5-39(a)Homologous recomnination with fransfected disruption constructs can inactivate specific target genes in yeast

Normal Yeast Genes Can Be Replaced with Mutant Alleles by Homologous Recombination

205 EXPERIMENTAL FIGURE 5-39(b)Homologous recomnination with fransfected disruption constructs can inactivate specific target genes in yeast

Study essential genes by conditional

knockout

Gal1 Promoter-Essential Gene

Grow in Galactose medium

Gal1 Promoter-Essential Gene

Grow in Glucose mediumMutant phenotype

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

206 Specific Genes Can Be Permanently Inactivated in the Germ Line of Mice

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

Video

Microinjection of ES cells into a blastocyst

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

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

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

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

Conditional Knockout: To Study Embryonic Lethal Essential Gene KO

208 EXPERIMENTAL FIGURE 5-42The loxP-Cre recombination system can knock out genes in specific cell types

Somatic Cell Recombination Can Inactivate Genes in Specific Tissues

Video

Creating a Transgenic Mouse

209 EXPERIMENTAL FIGURE 5-43Transgenic mice are produced by random integration of a foreign gene into the mouse germ

Dominant-Negative Alleles Can Functionally Inhibit Some Genes

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

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

RNA Interference Causes Gene Inactivation by Destroying the Corresponding mRNA

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

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

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

Review KEY CONCEPTS OF SECTION 5.5

Inactivating the Function of Specific Genes in Eukaryotes(p211)

Discussion: Answer Chapter 5 Questions

Homework: Review Chapter 5

1. Key Terms (p212)2. Concepts p212 (will be tested in Final)3. Analyzing the data p213-214 (These will be tested in Final)

Next Thursday Chapter 5 Test.

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

KEY WORDS