基因工程 Genetic Engineering 基因工程 Jacky Sun, Ph.D.. October 19, 2015October 19,...

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.


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


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.


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%


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


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


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


IsolationIsolation

Target sequence Target sequence

Interest Gene Interest Gene

PCR/CloningPCR/Cloning

Enzyme sitesEnzyme sites Vector Vector


2. Significance and Concerns

* Significance Significance * ConcernsConcerns


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.


1.Basic research on gene structure and function


2.Production of useful proteins by novel methods

prokaryotic

eucaryotic


3.Generation of transgenic plants and animals

Adversity 抗逆基因 gene Fluorescence 荧光 gene


Concerns担忧

1. The ethical 伦理的 problems


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.


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


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.


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


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.


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"


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


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


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++


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.


DNA Fragments Visualized Under UV Light


Southern Hybridization1


Southern Hybridization2


Northern Hybridization


Restriction Digestion

and Ligation


Cloning by Plasmid


Transformation

+

Plasmid DNA

Bacteria picks up a piece of DNA


PCR Reaction


Genomic PCR

1.5 kb

0.2 kb0.1 kb


DNA Sequencing


Automated DNA sequencing


Mutagenesis in vitro


6. Concept Map

基因工程 Genetic Engineering 基因工程 Jacky Sun, Ph.D.. October 19, 2015October 19,...

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