Molecular Cell Biology Chapter 5. Molecular Genetic Techniques Professor Dawei Li daweili@sjtu.edu.cn 3420-4744 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

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

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 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.

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

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

Replication Vector +DNA Fragment 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

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

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

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

179 FIGURE 5-14 DNA 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

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

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

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

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

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

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

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

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

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)

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

Dideoxy Sequencing of DNA Video Dideoxy Sequencing of DNA

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

Polymerase Chain Reaction Video Polymerase Chain Reaction

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

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

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

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 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 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)  

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

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

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

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

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

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

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

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

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

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)  

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

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

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

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

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

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

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  

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

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

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

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

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

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 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

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

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

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

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

207 EXPERIMENTAL FIGURE 5-41 ES 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

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

Creating a Transgenic Mouse Video Creating a Transgenic Mouse

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

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

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

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 Key Terms (p212) Concepts p212 (will be tested in Final) Analyzing the data p213-214 (These will be tested in Final) Next Thursday Chapter 5 Test.

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