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Genetic Engineering基因工程基因工程
Jacky Sun, Ph.D.Jacky Sun, Ph.D.
April 21, 2023April 21, 2023 Genetic EngineeringGenetic Engineering 22
Syllabus 课程提纲
CourseCourse 课程课程 :: Genetic EngineeringGenetic Engineering
Time:Time: Spring Semester, 2013Spring Semester, 2013
ReferencesReferences 参考参考 :: Gene VIII ( 基因八 [ 美 ] 莱威 · 本杰明 )
EssentialsEssentials 要素要素 of of Current 当代 Protocol in
Molecular Biology (Roger Brent, 2003)
Molecular Cloning: A Laboratory
Manual 指南指南 (Joseph Sambrook, 2001)
Instructor:Instructor: Guopeng Sun, Guopeng Sun, Ph.D.Ph.D.
April 21, 2023April 21, 2023 Genetic EngineeringGenetic Engineering 33
Objective1目标
1.1. Learn and understand some important Learn and understand some important conceptsconcepts 概念概念 and and definitionsdefinitions 定义定义 of of genetic engineering.genetic engineering.
2.2. Learn and understand some basic Learn and understand some basic methods used in methods used in recombinantrecombinant 重组重组 DNA DNA manipulationmanipulation 操作操作
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Objective2
3.3. Learn and understand what we can Learn and understand what we can do to use the theories and methods do to use the theories and methods of genetic engineering.of genetic engineering.
4.4. Learn and understand the standard Learn and understand the standard operation of the experiments that are operation of the experiments that are important methods in genetic important methods in genetic engineering.engineering.
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Grading评分
1.1. AttendanceAttendance 出勤出勤 : 10%: 10%
2.2. Experimental techniquesExperimental techniques :: 10%10%
3.3. Two Take-home TestsTwo Take-home Tests : : 10% 10%
4.4. Class Class participationparticipation 参与: 参与: 10%10%
5.5. Final exam: 60%Final exam: 60%
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Schedule进度表
节次 节次 1-2 Introduction 23-24 Using the genetic code
3-4 Genes are DNA 25-26 Protein synthesis
5-6 The content of the genome 27 Presentation for students
7-8 The replicon 5-13w Experiment (undetermined)
9-10 DNA replication
11-12 Recombination and repair
13-14 Transcription
15-16 The operon
17-18 Promoters and enhancers
19-20 RNA splicing and processing
21-22 Messenger RNA
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Chapter 1: Introduction1.1. Definition of Genetic EngineeringDefinition of Genetic Engineering
2.2. SignificanceSignificance 意义意义 and Concernsand Concerns
3.3. History and DevelopmentHistory and Development
4.4. MainstayMainstay of Genetic Manipulationof Genetic Manipulation
5.5. Common Techniques in Genetic Common Techniques in Genetic EngineeringEngineering
6.6. Concept MapConcept Map
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1. DefinitionGenetic EngineeringGenetic Engineering: tools and techniques ools and techniques used to used to isolateisolate 分离分离 one or more genes from one or more genes from an organism, an organism, modifymodify 修饰修饰 the material the material obtained, and obtained, and transfertransfer 转移转移 it into another it into another organismorganismRecombinant DNA technology: techniques of echniques of gene cloning.gene cloning.
Recombinant DNA:Recombinant DNA: the molecule formed by the molecule formed by joiningjoining 连接连接 a DNA of interest to vector DNAa DNA of interest to vector DNA
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IsolationIsolation
Target sequence Target sequence
Interest Gene Interest Gene
PCR/CloningPCR/Cloning
Enzyme sitesEnzyme sites Vector Vector
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2. Significance and Concerns
* Significance Significance * ConcernsConcerns
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Significance
1. Basic research on gene structure and function
2. Production of useful proteins by novel 新颖的 methods
3. Generation of transgenic plants and animals
4. Medical diagnosis 诊断 and treatment.
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1.Basic research on gene structure and function
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2.Production of useful proteins by novel methods
prokaryotic
eucaryotic
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3.Generation of transgenic plants and animals
Adversity 抗逆基因 gene Fluorescence 荧光 gene
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Concerns担忧
1. The ethical 伦理的 problems
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Concerns担忧
2. The problems of Genetically modified foods
Some research confirmed that the GM food could cause infertility 不孕 in mouse. However, there was no evidence that could happened to human.
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3. History and Early Development
*Three Main Three Main ErasEras 时代时代 of of Development of GeneticsDevelopment of Genetics
*Early Development of Gene Early Development of Gene CloningCloning
April 21, 2023April 21, 2023 Genetic EngineeringGenetic Engineering 1919
Three Main Eras of Development of
Genetics1
1. Rediscovery of Gregor Mendel‘s work in early 1900, elucidation 阐明 of the inheritance 遗传principles 原理 and
genetic mapping in the next 40 years.
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Three Main Eras of Development of Genetics2
2. Establishment 确立 of microbial genetics in mid- 1940s, role of DNA as the genetic material was confirmed 证实 , understanding the mechanisms 机制 of
gene transfer between bacteria
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Three Main Eras 时代 of Development of Genetics3
3. Discovery of the structure of DNA by James Watson and Francis Crick in 1953 leads genetics to molecular level, establishment of the complete genetic code in 1966 - the stage was now set for the appearance 出现 of the new genetics.
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The Nobel Prize in Physiology or Medicine 1962
Francis H. C. Crick James D. Watson Maurice H. F. Wilkins
"for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material"
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Early Development of Gene Cloning
1. Molecular glue 胶水 - DNA ligase isolated in 1967
2. Molecular scissors 剪刀 - the first restriction 限制性 enzyme isolated in 1970
3. First recombinant DNA molecules 分子generated at Stanford University in 1972
4. Joining DNA fragments 片段 to the plasmid 质粒 pSC101 in 1973
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4. Mainstay 中流砥柱 of Genetic Manipulation
((Discussion in classDiscussion in class)) TargetTarget 目标目标 Gene cloning: Single DNA fragment Gene cloning: Single DNA fragment
isolation; the genomic or cDNA isolation; the genomic or cDNA librarylibrary 文库文库constructionconstruction 建造建造 and and screeningscreening 筛选筛选 ; PCR; PCR
Recombinant DNA construction or Recombinant DNA construction or modificationmodification修饰修饰 in in vitrovitro 体外体外 :: restriction enzymes, restriction enzymes, modifying enzymes, vectors; PCRmodifying enzymes, vectors; PCR
DNA DNA amplificationamplification 扩增扩增 in in vivovivo 体内体内 :: transformationtransformation 转化转化 and selection and selection in vitroin vitro; ; Research and application: recombinant protein Research and application: recombinant protein expression, transgenic, cloning, medical expression, transgenic, cloning, medical diagnosisdiagnosis 诊断诊断 , gene therapy, gene therapy
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April 21, 2023April 21, 2023 Genetic EngineeringGenetic Engineering 2626
5. Common Techniques* Southern Hybridization 杂交* Northern Hybridization* PCRPCR++* Restriction digestion Restriction digestion ++* DNADNA sequencingsequencing* DNA cloning and Transformation DNA cloning and Transformation ++* Mutagenesis Mutagenesis in vitroin vitro++
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Aragrose Gel Electrophoresis to Separate DNA
Agarose is an inert 惰性惰性 carbohydrate 碳水化合物碳水化合物isolated from seaweeds. DNA molecules in the gel can be visualized 直观直观 by staining 染色染色 with EB and observe under UV light.
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DNA Fragments Visualized Under UV Light
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Southern Hybridization1
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Southern Hybridization2
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Northern Hybridization
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Restriction Digestion
and Ligation
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Cloning by Plasmid
April 21, 2023April 21, 2023 Genetic EngineeringGenetic Engineering 3434
Transformation
+
Plasmid DNA
Bacteria picks up a piece of DNA
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PCR Reaction
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Genomic PCR
1.5 kb
0.2 kb0.1 kb
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DNA Sequencing
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Automated DNA sequencing
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Mutagenesis in vitro
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6. Concept Map