医用分子遗传学 本课程主要包括分子遗传学的基本原理以及现代基因 工程和蛋白质工程及其在医学中的应用。 要求同学能够掌握基因表达与调控、基因工程和蛋白 质工程、基因诊断和基因治疗的基本理论和方法。 考核方式：闭卷考试 负责教师：于 敏（ 魏欢欢（ ） 教科书：医用分子遗传学（复旦大学出版社） 教学参考书： Robert F. Weaver 分子生物学 （ Molecular Biology ）科学出版社（ McGraw-Hill ）

第一章 重组 DNA 技术－基因工程 及其与医学的关系 复旦大学分子医学教育部重点实验室 于 敏

Warming up DNA Recombination Gene Engineering Application of Gene Engineering in Medical Science

Warming up - Basic facts of RNA and DNA

Single Strand RNA

Single Strand DNA

DNA 的四种碱基相互配对

Double Strand DNA

Central Dogma of Molecular Biology Reverse Transcription

DNA and Protein Electrophoresis Agarose gel electrophoresis Polyacrylamide gel electrophoresis (PAGE)

12 Gel Electrophoresis A technique used to separate DNA fragments by size The gel (agarose or polyacrylamide) is subjected to an electrical field The DNA, which is negatively-charged, migrates towards the positive pole The larger the DNA fragment, the slower it will move through the gel matrix DNA is visualized using fluorescent dyes

Protein Electrophoresis

DNA cloning Applications in enzymology Hybridization DNA Sequencing Polymerase Chain Reaction （ PCR ） I. Recombinant DNA cloning technology

1. DNA cloning To obtain large amounts of pure DNA Procedure Isolate DNA Use restriction enzymes to cut DNA Ligate fragments into a cloning vector Transform recombinant DNA into a host to replicate the DNA and pass copies into progeny.

DNA Manipulation The molecular biology revolution started with the discovery of restriction endonucleases Enzymes that cleave DNA at specific sites These enzymes are significant in two ways 1. Allow a form of physical mapping that was previously impossible 2. Allow the creation of recombinant DNA molecules (from two different sources)

Molecular Cloning A clone refers to a genetically identical copy Molecular cloning is the isolation of a specific DNA sequence (usually protein-encoding) Sometimes called gene cloning The most flexible and common host for cloning is E. coli Propagation of DNA in a host cell requires a vector

Vectors Plasmids are small, circular extrachromosomal DNA molecules Used for cloning small pieces of DNA Have three important components 1. Origin of replication 2. Selectable marker 3. Multiple cloning site (MCS)

Vectors

Phage vectors are modified bacterial viruses Most based on phage lambda (l) of E. coli Used to clone inserts up to 40 Kbp Have two features not shared with plasmid vectors They kill their host cells They have linear genomes Middle replaced with inserted DNA

Vectors

Restriction endonucleases DNA polymerase Ⅰ Reverse transcriptase DNA ligase Alkaline phosphatase Terminal transferase Taq DNA polymerase 2. Applications in enzymology

It can recognize special sequences and cleave DNA at these specific base sequences. Type II can recognize palindrome sequences. Restriction endonuclease GGATCC CCTAGG

Palindrome Palindrome is also called inverted repeat sequence, which means the nucleotide sequence in 5′to 3′direction is the same in both strands.

Palindrome （ 回文序列） 客上天然居， 居然天上客。 僧游云隐寺， 寺隐云游僧 。 Madam, I'm Adam. Was it a car or a cat I saw?

Restriction Enzymes Recognize a specific DNA sequence (restriction site) Break a phosphodiester linkage between a 3’ carbon and phosphate Used for Create DNA fragments for cloning Analyze positions of restriction sites in cloned or genomic DNA

Restriction Enzymes Are found naturally in bacteria as a defense against vital DNA. Restriction sites are methylated in bacteria, and thus protected. Are denoted by three letter names derived from the bacterial strain they originate from.

Restriction Enzymes: EcoRI EcoRI (“Echo R one”) is a commonly used enzyme. It was the first (one) restriction enzyme isolated from the “R” strain of E. coli. It demonstrates the usual type recognition site, a palindrome (the same on both strands, reading in opposite directions) EcoRI leaves a four base, 5’ overhang, sticky end.

Restriction Enzyme Sites Sma I (from Serratia marcescens) cuts a palindrome to give blunt ends. BamHI (from Bacillus amyloliquefaciens H) cuts to give a 5’ overhang. PstI (from Providencia stuartii) cuts to give a 3’ overhang.

Restriction Enzymes Blunt ends: both strands are cut at the same position. Sticky ends: overhanging regions (3’ or 5’) are useful in cloning. They are complementary, thus anneal, DNA ligase can covalently link them.

Restriction Enzyme Analysis of Cloned DNA sequences Cloned DNA can be cut with restriction enzymes and electrophoresed on agarose gels and visualized with ethidium bromide, in order to map its restriction sites DNA cut with several enzymes, each loaded in a lane of an agarose gel.

Restriction enzyme analysis of cloned DNA sequences DNA is stained with ethidium bromide, which fluoresces under UV when complexed with DNA. The gel is photographed, and the distance migrated by each band of identical DNA molecules is measured and compared with a calibration curve. Restriction mapping may be done with a circular plasmid, a cloned sequence, or a fragment of plasmid prepared by gel cutting.

Recombination of DNA

Ligation of DNA fragments

Ligation of DNA Ligation of sticky end DNA ligase Ligation of blunt ends DNA ligase Alkaline phosphatase The addition of a homopolymer tail DNA ligase Terminal transferase

3. DNA Hybridization

Membrane hybridization assay

In situ hybridization

Southern and Northern blot

Radiolabeling of DNA

4. DNA Sequencing Dideoxy Method Automated DNA Sequencing

Dideoxyribonnucleotide

5. Polymerase Chain Reaction The polymerase chain reaction (PCR) is a rapid and versatile in vitro method for amplifying DNA.

PCR reaction system DNA template A pair of primers DNA polymerase (Taq) dNTPs Mg 2+ -containing buffer

Procedures of PCR Denaturing: the template DNA is denatured to become ssDNA from dsDNA by heating. Annealing: this step allows the hybridization of the primers with target DNA. Extension: this process is the DNA synthesis step.

ing

The first three cycles of PCR