上海交通大学：《微生物学 Microbiology》课程教学资源（双语课件）Lecture 11.1 Genetic Engineering（Methods for Manipulating DNA）

上泽充通大￥ Shanghai Jiao Tong University 1896 1920 1987 2006 m Lecture 11-1 Genetic Engineering Chapter 11 in BROCK BIOLOGY OF MICROORGANISMS NG UNI Chen Feng School of Life Science and Technology, Shanghai Jiao Tong University http://micro.sjtu.edu.cn

1896 1920 1987 2006 Lecture 11-1 Genetic Engineering Chapter 11 in BROCK BIOLOGY OF MICROORGANISMS Chen Feng School of Life Science and Technology, Shanghai Jiao Tong University http://micro.sjtu.edu.cn

I.Methods for Manipulating DNA 11.1 Restriction and Modification Enzymes Genetic engineering:using in vitro techniques to alter genetic material in the laboratory Basic techniques include Restriction enzymes ·Gel electrophoresis Nucleic acid hybridization ·Nucleic acid probes ·Molecular cloning ·Cloning vectors Chen Feng,Lecture of Microbiology

Chen Feng, Lecture of Microbiology I. Methods for Manipulating DNA 11.1 Restriction and Modification Enzymes Genetic engineering: using in vitro techniques to alter genetic material in the laboratory • Basic techniques include • Restriction enzymes • Gel electrophoresis • Nucleic acid hybridization • Nucleic acid probes • Molecular cloning • Cloning vectors

Restriction Enzymes Restriction enzymes:recognize specific DNA sequences and cut DNA at those sites Widespread among prokaryotes ·Rare in eukaryotes Protect prokaryotes from hostile foreign DNA (e.g.,viral genomes) e Essential for in vitro DNA manipulation Chen Feng,Lecture of Microbiology

Chen Feng, Lecture of Microbiology Restriction Enzymes Restriction enzymes: recognize specific DNA sequences and cut DNA at those sites • Widespread among prokaryotes • Rare in eukaryotes • Protect prokaryotes from hostile foreign DNA (e.g., viral genomes) • Essential for in vitro DNA manipulation

Restriction Enzymes Three classes of restriction enzymes Type ll cleave DNA within their recognition sequence and are most useful for specific DNA manipulation(Figure 11.1a) Restriction enzymes recognize inverted repeat sequences(palindromes) Typically 4-8 base pairs long;EcoRl recognizes a 6-base- pair sequence Sticky ends or blunt ends Chen Feng,Lecture of Microbiology

Chen Feng, Lecture of Microbiology Three classes of restriction enzymes • Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation (Figure 11.1a) Restriction enzymes recognize inverted repeat sequences (palindromes) • Typically 4–8 base pairs long; EcoRI recognizes a 6-base￾pair sequence Sticky ends or blunt ends Restriction Enzymes

Figure 11.1a 5' G-A-A-T-T-C 3' 3' C-T-T-A-A-G 5' A-A-T-T- 5' -G 3' 3' -C-T-T-A-A. G- 5' Single-stranded (a) “sticky”ends 2012 Pearson Education,Inc

Figure 11.1a Single-stranded “sticky” ends © 2012 Pearson Education, Inc

Gel electrophoresis Gel electrophoresis:separates DNA molecules based on size Electrophoresis uses an electrical field to separate charged molecules 。 Gels are usually made of agarose,a polysaccharide 0 Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups Gels can be stained with ethidium bromide and DNA can be visualized under UV light Chen Feng,Lecture of Microbiology

Chen Feng, Lecture of Microbiology Gel electrophoresis Gel electrophoresis: separates DNA molecules based on size • Electrophoresis uses an electrical field to separate charged molecules • Gels are usually made of agarose, a polysaccharide • Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups Gels can be stained with ethidium bromide and DNA can be visualized under UV light

Figure 11.2 A B D Size in base pairs 5000 4000一 3000一 2000- 1800- 1000一 500- + (a) () 2012 Pearson Education,Inc 2012 Pearson Education,Inc

Figure 11.2 © 2012 Pearson Education, Inc

11.2 Nucleic Acid Hybridization Nucleic acid hybridization:base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a nucleic acid probe Southern blot:a hybridization procedure where DNA is in the gel and probe is RNA or DNA Northern blot:RNA is in the gel Chen Feng,Lecture of Microbiology

Chen Feng, Lecture of Microbiology 11.2 Nucleic Acid Hybridization Nucleic acid hybridization: base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix • Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a nucleic acid probe Southern blot: a hybridization procedure where DNA is in the gel and probe is RNA or DNA • Northern blot: RNA is in the gel

Figure 11.4 123456123456 二 2012 Pearson Education,Inc

Figure 11.4 © 2012 Pearson Education, Inc

11.3 Essentials of Molecular Cloning Molecular cloning:isolation and incorporation of a piece of DNA into a vector so it can Foreign DNA be replicated and manipulated Cut with restriction enzyme Three main steps of gene Add vector cut Sticky with same ends restriction enzyme cloning: Vector Add DNA ligase to 1.Isolation and fragmentation form recombinant molecules of source DNA Cloned- DNA 2. Insertion of DNA fragment Introduction of recombinant into cloning vector vector into a host Puaron Eduption ine 3.Introduction of cloned DNA into host organism Chen Feng,Lecture of Microbiology

Chen Feng, Lecture of Microbiology 11.3 Essentials of Molecular Cloning Molecular cloning: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated Three main steps of gene cloning: 1. Isolation and fragmentation of source DNA 2. Insertion of DNA fragment into cloning vector 3. Introduction of cloned DNA into host organism