1. Molecular Biology Fourth Edition
Lecture PowerPoint to accompany
Molecular Biology Fourth Edition
Robert F. Weaver
Chapter 4
Molecular Cloning Methods
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. Chapter 4
Molecular Cloning Methods
Page 48-70

3. 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 cloning
Separate a gene from the other genes
Amplification of modified forms of genetic materials
Manipulation of a piece of DNA for further experiments
Vector (DNA carrier)
Plasmids
Cosmids
YAC
Bateriophage
Virus

4. Restriction Enzymes that can cut DNA
Enzymes(ligase) that can join DNA

5. Restriction Endonucleases(RE) --The Molecular Scissors
Host enzymes that prevent the invasion of foreign DNAs such as viral DNA, by cutting them up.
Restriction
These enzymes cut within the foreign DNAs, rather than chewing them away from the ends.
Endonucleases
These enzymes recognize a specific DNA sequence (4-12bp) which is twofold symmetry and cut both DNA strands
Palindrome
Some enzymes make staggered cuts
GAATTC
CTTAAG
Some make even cuts
CCCGGG
GGGCCC

6. Table 4.1
Isoschizomer
Heteroschizomer
neoschizomer

7. S -- deoxyribose P -- phosphate groups 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’

8. Sticky end
ligation

10. Figure 4.1 제한효소는 왜 자신의 DNA는 절단하지 않는가?
Restriction-Modification system
=R-M System

11. Vectors -- the DNA carriers
DNA Carrier: Capable of replicating in bacteria
Allow the vector as well as the foreign DNA to amplify in the host cell
Plasmids
1. Origin of replication
3.Multiple cloning sites
2.Antibiotic-resistant genes
4. Circle form
(supercoiled form)

12. 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

13. Recombinant DNA 재조합 DNA
The first cloning experiment done by Boyer and Cohen 참고
r: resistant
s: susceptible
Recombinant DNA
재조합 DNA

14. A
Screen
Selection
A region이 MCS 라면?
Further study: replica

15. Self ligation 방지 1) Alkaline phosphatase 2) Directional cloning
smaI
2) One cutting selection
: blunt end
1000bp
smaI
smaI
200bp
Figure 4.3
EcoRI

16. Transformation
Cloning된 DNA를 E.coli에 도입하는 방법
1) CaCl2 - Heat shock
2) Electroporation

17. Transformation (heat shock method)

18. Transformation (heat shock method)

19. Amps
AmpR – recombinant DNA OK!

20. White and blue selection with LacZ selection marker.
Encoded by LacZ gene

21. White and blue selection with LacZ selection marker.
Multiple cloning sites
LacZ gene
(b-galactosidase)

22. White and blue selection with LacZ selection marker.

23. White and blue selection with LacZ selection marker.
X-gal
X-gal

24. +
X-gal
X-gal
AMPR

25. Lac operon의 원리 Inducer: IPTG 그림 4.7 pBluescript 벡터.
그림 4.7 pBluescript 벡터. 이 플라스미드는 pBR322를 기본으로 하고, 그 벡터
의 앰피실린 저항성 유전자(초록색)와 복제기점(보라색)을 가지고 있다. 더구나 파지
f1의 복제기점(주황색)을 가지고 있어 세포가 복제기구를 제공하고, f1 도움 파지에
의해 감염되면 단일가닥 형태로 분비될 수 있다. 다중클로닝 부위(MCS, 빨간색)는
2개의 파지 RNA 중합효소 프로모터(T7과 T3) 사이에 21개의 독특한 제한부위를
가지고 있다. 그러므로 어떤 DNA 삽입체를 파지 RNA 중합효소에 의해 시험관 내에
서 전사시키면 두 가닥의 RNA를 얻을 수 있다. 다중클로닝 부위는 대장균 lacZ ′ 유
전자(연한 파란색) 안에 삽입되어 있다. 그러므로 플라스미드가 절단되지 않으면
IPTG와 같은 유도자를 첨가했을 때 lacI 유전자(노란색)에 의해 만들어지는 억제인
자의 기능을 방해하고, 이에 따라 β-갈락토시데이즈 N-말단 절편을 생성할 것이다.
그러므로 절단되지 않은 벡터를 갖는 클론은 지시자 X-gal을 첨가하면 파란색으로
변할 것이다. 반대로 다중제한 부위에 삽입체가 있는 재조합 플라스미드를 갖는 클론
은 lacZ ′ 유전자가 절단되어 기능적인 β-갈락토시데이즈를 만들 수 없을 것이다. 그
러므로 이러한 클론은 흰색으로 남는다.
Lac operon의 원리
Inducer: IPTG
그림 4.7 pBluescript 벡터.

26. PCR -- polymerase chain reaction
Page 59
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).

27. PCR -- polymerase chain reaction을 위한 주요 STEP 3
Forward primer
Reverse primer
Taq DNA polymerase

28. Figure 4.11
증폭(amplification)
Thermal cycler

29. Heat Heat The ideal PCR products 1st cycle 2nd cycle 5’ 3’ 5’ 3’ 5’ 3’

30. Heat 3rd cycle The ideal PCR products 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’

31. 2n products Heat Heat 4th cycle 5th cycle 6th cycle nth cycle 5’ 3’ 5’

33. Human insulin CDS Q: PCR primer 작성해 보기
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

34. cDNA 클로닝 Page 57 Human genomic library Gene (DNA) RNA protein Gene
transcription
Protein
Protein
translation
Protein activities

35. The Central Dogma DNA Precursor RNA mRNA Protein exon intron cDNA
Double-stranded
intron
Precursor
RNA
AAAAAAAAAAn
AAAAAAAAAAn
Single-stranded
mRNA
AAAAAAAAAAn
double-stranded
Reverse transcription
Protein
cDNA

36. 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 make
a protein

37. Single strand DNA Break
cDNA synthesis
TTTTTTTTT
Nick translation
Single strand DNA Break
First strand
cDNA library

38. Protein Expression cDNA Human genomic library Gene (DNA) Gene RNA
transcription
translation
Gene
Protein
Protein activities

39. Genomic library cDNA library Source Variation Insert size
Genomic DNA
mRNA
Source
Species or strains
Tissues
Developmental stages
Variation
12k -- 20k
0.2k -- 6k
Insert size
Equal
Correlate with
expression level
Representation
Gene structure
Infer protein identity
Encoded protein
Purpose

40. Real Time (RT)-PCR 원리
F: Fluorescence
Q:Quenching
그림 4.17

41. Real Time (RT)-PCR 기기

42. Real Time (RT)-PCR 분석

43. Real Time (RT)-PCR 분석
CT: Cycle Threshold

44. 4.3 클론된 유전자의 발현방법
Figure 4.17

45. Expression vectors (발현 벡터)
To produce the product of a cloned gene for further studies
Expression vectors with a strong promoter & 리보솜 결합부위
More mRNA
More protein
Expression vectors with an inducible promoter
Foreign proteins when overexpressed could be toxic
Keep the gene expression off till it is time to turn it on
Drug-inducible (e.g. IPTG or arabinose)
Heat-inducible
3) Expression vectors with a fusion tag for affinity purification
Facilitate the purification of the expressed protein
6 Histidine tag
Glutathione transferase tag (GST)
Maltose-binding protein tag

46. + LacI + LacZ
Figure 4.16

47. 그림 4.15 PBAD 벡터 사용. 그림 4.15 PBAD 벡터 사용. 초록색 형광단백질(GFP) 유전자가 PBAD 프로모터에
의하여 조절되도록 클로닝하였고 프로모터의 활성은 아라비노즈의 농도 증가에 의
해 유도되었다. 사진 위에 농도가 표시된 만큼의 아라비노즈가 주어진 세포에서 얻
은 단백질을 전기영동 막에 블롯팅한 후 항체를 이용하여 발현된 GFP 단백질을 검
출하였다(면역블롯팅, 5장 참조). (출처: Copyright 2003 Invitrogen Corporation. All Rights
Reserved. Used with permission.)
그림 4.15 PBAD 벡터 사용.

48. Figure 4.16 올리고-히스티딘 발현벡터의 이용법

49. 진핵 세포 발현 시스템 목적
셔틀 벡터
Transfection: Ca3(PO4)2, Liposome, Viral infection