医用分子遗传学
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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（minyu@shmu.edu.cn)
魏欢欢（weiyh@fudan.edu.cn ）
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
2011.9.
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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 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
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Agarose gel electrophoresis
Polyacrylamide gel electrophoresis
(PAGE)
Gel Electrophoresis
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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
12
Protein Electrophoresis
I. Recombinant DNA
cloning technology
DNA cloning
Applications in enzymology
Hybridization
DNA Sequencing
Polymerase Chain Reaction（PCR ）
1. DNA cloning
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To obtain large amounts of pure DNA
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Procedure
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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)
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Molecular Cloning
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A clone refers to a genetically identical copy
Molecular cloning is the isolation of a specific
DNA sequence (usually protein-encoding)
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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
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Plasmids are small, circular extrachromosomal
DNA molecules
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Used for cloning small pieces of DNA
Have three important components
1. Origin of replication
2. Selectable marker
3. Multiple cloning site (MCS)
Vectors
Vectors
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Phage vectors are modified bacterial viruses
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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
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They kill their host cells
They have linear genomes
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Middle replaced with inserted DNA
Vectors
2. Applications in enzymology
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Restriction endonucleases
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DNA polymeraseⅠ
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Reverse transcriptase
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DNA ligase
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Alkaline phosphatase
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Terminal transferase
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Taq DNA polymerase
Restriction endonuclease
It can recognize special sequences and
cleave DNA at these specific base
sequences.
Type II can recognize palindrome
sequences.
GGATCC
CCTAGG
Palindrome
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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
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Recognize a specific DNA sequence
(restriction site)
Break a phosphodiester linkage between a 3’
carbon and phosphate
Used for
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Create DNA fragments for cloning
Analyze positions of restriction sites in cloned
or genomic DNA
Restriction Enzymes
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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
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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
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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
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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
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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
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DNA template
A pair of primers
DNA polymerase (Taq)
dNTPs
Mg2+-containing buffer
Procedures of PCR
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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