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Molecular BiologyFourth Edition
Chapter 4
Molecular Cloning Methods
Lecture PowerPoint to accompany
Robert F. Weaver
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Page 48-70
Molecular Cloning Methods
Chapter 4
Molecular Cloning
The process of inserting a piece of DNA molecule of interest into a DNA carrier (vector) in order to make multiple copies of the DNA of interest in a host cell such as bacteria
Purposes of molecular cloningSeparate a gene from the other genesAmplification of modified forms of genetic materialsManipulation of a piece of DNA for further experiments
Vector (DNA carrier)•Plasmids•Cosmids•YAC•Bateriophage•Virus
Restriction Enzymes that can cut DNA
Enzymes(ligase) that can join DNA
Restriction Endonucleases(RE)--The Molecular Scissors
Host enzymes that prevent the invasion of foreign DNAs such as viral DNA, by cutting them up.
These enzymes cut within the foreign DNAs, rather than chewing them away from the ends.
Restriction
Endonucleases
These enzymes recognize a specific DNA sequence (4-12bp) which is twofold symmetry and cut both DNA strands
Some enzymes make staggered cutsGAATTCCTTAAG
Some make even cuts CCCGGGGGGCCC
Palindrome
Table 4.1
IsoschizomerHeteroschizomerneoschizomer
S -- deoxyribose P -- phosphate groups
3’
3’
5’
5’
5’ 3’
3’5’
5’ 3’
3’ 5’
Sticky end
Sticky end
ligation
Figure 4.1제한효소는 왜 자신의 DNA 는 절단하지 않는가 ?
Restriction-Modification system=R-M System
Vectors -- the DNA carriersDNA Carrier: Capable of replicating in bacteriaAllow the vector as well as the foreign DNA to amplify in the host cell
1) Plasmids
1. Origin of replication
2.Antibiotic-resistant genes
3.Multiple cloning sites
4. Circle form (supercoiled form)
Vectors -- the DNA carriers
• Plasmid as a vector
• Host: E. coli
• Vector size: usually about 3kb.
• Insert size: up to 20kb. usually below 5 kb.
• Insert select: functional activation of the ability
to resist an antibiotics
The first cloning experiment done by Boyer and Cohen
Recombinant DNA재조합 DNA
참고r: resistants: susceptible
• Screen• Selection
Further study: replica
A region 이 MCS 라면 ?
A
Figure 4.3
2) Directional cloning
Self ligation 방지1) Alkaline phosphatase
2) One cutting selection: blunt end
EcoRI
1000bp
200bp
smaI
smaI smaI
Transformation
Cloning 된 DNA 를 E.coli 에 도입하는 방법1) CaCl2 - Heat shock
2) Electroporation
Transformation(heat shock method)
Transformation(heat shock method)
Amps AmpR – recombinant DNA OK!
White and blue selection with LacZ selection marker.
Encoded by LacZ gene
LacZ gene(-galactosidase)
Multiple cloning sites
White and blue selection with LacZ selection marker.
White and blue selection with LacZ selection marker.
X-gal
X-gal
White and blue selection with LacZ selection marker.
X-gal
X-gal
+
AMPR
그림 4.7 pBluescript 벡터 .
Lac operon 의 원리Inducer: IPTG
• PCR -- polymerase chain reaction• 중합 효소 연쇄 반응
• PCR is one of the most powerful molecular biology techniques.
• It allows scientists to amplify a specific DNA region in the test tube from extremely tiny amount of DNA sample
• (even from a single molecule of DNA).
Page 59
PCR -- polymerase chain reaction 을 위한 주요 STEP 3
Taq DNA polymerase
Forward primer
Reverse primer
Figure 4.11
증폭 (amplification)
Thermal cycler
5’ 3’
5’3’
5’
5’ 3’
3’
5’
5’
5’
5’
Heat1st cycle
Heat2nd cycle
5’ 3’5’3’
5’
3’
5’ 3’
5’
3’
3’ 5’
3’
3’
3’
3’
The ideal PCR products
Heat3rd cycle
The ideal PCR products
5’
5’
5’
5’
5’
3’
5’ 3’
5’
3’
3’ 5’
3’
3’
3’
3’
3’ 5’
3’5’
5’3’
3’5’
5’3’
5’3’
5’3’
3’5’
5’ 3’
3’5’
3’5’
3’ 5’
3’ 5’
3’
5’ 3’
5’3’
5’
5’3’3’5’
Heat4th cycle
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
Heat5th cycle
Heat6th cycle
Heatnth cycle
5’3’3’5’
2n products
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’3’3’5’
5’ 3’5’3’
5’
5’
Human insulin CDS
5’----Atg gcc ctg tgg atg cgc ctc ctg ccc ctg ctg gcg ctg ctg gcc ctc tgg gga cct gac cca gcc gca gcc ttt gtg aac caa cac ctg tgc ggc tca cac ctg gtg gaa gct ctc tac cta gtg tgc ggg gaa cga ggc ttc ttc tac aca ccc aag acc cgc cgg gag gca gag gac ctg cag gtg ggg cag gtg gag ctg ggc ggg ggc cct ggt gca ggc agc ctg cag ccc ttg gcc ctg gag ggg tcc ctg cag aag cgt ggc att gtg gaa caa tgc tgt acc agc atc tgc tcc ctc tac cag ctg gag aac tac tgc aac tag—3’
•translation="MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN"
Q: PCR primer 작성해 보기
Human genomic library
Gene
Protein
Protein activities
Protein
Gene (DNA)
RNA
protein
transcription
translation
Page 57 cDNA 클로닝
The Central Dogma
DNA
mRNA
Protein
Double-stranded
Precursor RNA
exon
intron
AAAAAAAAAAn
AAAAAAAAAAn Single-stranded
AAAAAAAAAAn
double-stranded
Reverse transcription
cDNA
cDNA
A cDNA or complementary DNA, is a copy DNA of an RNA, usually mRNA
cDNA mRNA
Double stranded Single stranded
Stable unstable
Easy to manipulate More difficult to manipulate
Need to be transcribed into RNA to make a protein
Can be directly used to makea protein
cDNA synthesisTTTTTTTTT
First strand Nick translationSingle strand DNA Break
cDNA library
Protein Expression
Human genomic library
Gene
Protein
Protein activities
Gene (DNA)
RNA
protein
transcription
translation
cDNA
Genomic library cDNA library
Genomic DNA mRNASource
Species or strains Species or strainsTissuesDevelopmental stages
Variation
12k -- 20k 0.2k -- 6kInsert size
Equal Correlate with expression level
Representation
Gene structureInfer protein identity
Encoded proteinInfer protein identity
Purpose
그림 4.17
Real Time (RT)-PCR 원리
F: FluorescenceQ:Quenching
Real Time (RT)-PCR 기기
Real Time (RT)-PCR 분석
Real Time (RT)-PCR 분석
CT: Cycle Threshold
Figure 4.17
4.3 클론된 유전자의 발현방법
Expression vectors ( 발현 벡터 )
To produce the product of a cloned gene for further studies
1) Expression vectors with a strong promoter & 리보솜 결합부위
More mRNA More protein
2) Expression vectors with an inducible promoter
Foreign proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG or arabinose)b. Heat-inducible
3) Expression vectors with a fusion tag for affinity purificationFacilitate the purification of the expressed protein
1) 6 Histidine tag2) Glutathione transferase tag (GST)3) Maltose-binding protein tag
Figure 4.16
+ LacI + LacZ
그림 4.15 PBAD 벡터 사용 .
Figure 4.16올리고 - 히스티딘 발현벡터의
이용법
진핵 세포 발현 시스템
• 목적• 셔틀 벡터• Transfection: Ca3(PO4)2, Liposome, Viral
infection
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