DNA technology (DNA 조작기술 ) 유전자 cloning Cloning & Plasmid Joshua Ledergerg & Edward Tatum, 1946 두종류의다른유전자를가진대장균사이에유전자의재조합이일어나새로운대장균이만들어지는것을발견했다. 이원리를바탕으로 1970 년대에 DNA 재조합기술이발전하게되었다. 원핵생물에서 DNA 의이동방법 플라스미드를이용한 cloning 1. 형질전환 (Transformation) 세포주변에있는유전물질을받아들이는방법 (Frederick Griffith 1920) 2. 형질도입 (Transduction) 세균의유전자를박테리오파지가전달 3. 접합 (Conjugaton) 두개의세포사이에접합을통한 DNA 이동 1. 미생물로부터플라스미드를분리한다. 2. 동물, 식물로부터특정한유전자가포함된 DNA를분리한다. 3. 분리한유전자를포함하고있는 DNA조각을플라스미드에삽입하여재조합 DNA를만든다. 4. 박테리아세포에재조합플라스미드를넣어형질전환을한다. 5. 재조합박테리아클론이확보사용된다. Plasmid selection selectable markers - plasmid 삽입된 cell 과삽입되지않는 cell 구별을위해사용 - antibiotic resistance 이용 ; plasmid 삽입된 cell 만항생제함유배지에서생존 - antibiotic resistance chemical modification 통해서 target antibiotics inactivation 세포막을통한 antibiotics transport 방해 DNA Recombination inserting new genes into plasmids - gene cloning technology ; cut and past DNA fragment - plasmid vector 는 cloning site( 제한효소인식서열 ) 포함 ; 원형의 plasmid 절단하여 open, DNA 삽입가능 - 제한효소 sticky end 형성 ; 보완적인다른 fragment 와수소결합형성하므로 DNA ligase 를위해충분한시간동안 DNA fragment 잡아둠 - DNA ligase ; 인접한 nucleotide 사이에 phosphodiester 결합재형성 stable double helix 1
The host cell : Escherichia Coli Transformation E. Coli DNA 증식을위해 transformation 중요 Transformation을위해요구되는요소 - gene 도입을위한적당한숙주 - 숙주안으로 gene을도입할운반체 -gene을받아들인숙주선별할수단 Bacterium E. coli - 가장넓게사용 ; simple, genetic environment 잘알려짐 - genome 완전히분석됨 - genetic code 보편적이기때문에다른생물의외래 DNA 받아들임 DNA의구성, 구조, 기본 mechanism 동일하므로복제가능 - 빠르게분열하고, cell 분열할때마다도입된 DNA도복제 - culture medium에서 37 일때최고성장 - bacterial growth ; CaCl2 + heat shock(42 ) 조건에서형질전환일어남 다른이온 (Mg2+, Mn2+,Ba2+ 등 ) 도사용 ; mixture of positive ion 사용시 효율증가 DNA size 와 conformation 이형질전환효율에영향 A subset of cell 에제한받음 plasmid 수증가해도형질전환된 cell 수변화없음 E. coli 가 DNA 받아들이는정확한메커니즘밝혀지지않음 adhesion zone 가설 ; 세포막의 adhesion zone 에서 channel 형성단점 ; Large DNA 는성공적으로형질전환되기힘듦 Proposed molecular mechanism of DNA transformation of E. coli Directional cloning 0 처리하여세포막고형화, charged phosphate stabilizing - Transformation solution속의양이온들이 phosphate group과 complex 이룸 (-) charge 가리므로 DNA 분자이동가능 - Heat shock 세포막의열적불균형형성하여 adhesion zone 통한 DNA pumping 도움 Genomic library ( 유전자도서관 ) 특정유전자를갖고있는클론을찾는방법 무차별유전자클로닝방법 1. 제한효소를이용하여 DNA 를수천조각으로절단 2. 각 DNA 조작은서로다른벡터분자에실려박테리아세포에형질전환 3. 수많은종류의박테리아클론을 genomic library 라함 Probe 찿고자하는유전자 X 가 TAGGCT 라는염기서열을가지고있다면, 방사선동위원소로표지후특정유전자발견하는데사용 Hybridization 탐지자가준비되면, 클론으로부터단일가닥의 DNA 를준비하며, 탐지자와 DNA 표본을혼합하면방사능을가진 RNA probe 가상보적인서열을가진 DNA 에수소결합을함으로서클론을표지한다. 2
Single stranded (SS) DNA can pair with a complementary strand to regenerate DS DNA Southern Blotting DNA fragments separated in a gel can be transferred to a membrane for hybridization to a SS DNA Prob. The extent of hybridization can be quantitated by using a radioactive DNA probe and auto-radiography The plasmid vector propagation of plasmids - bacterial cell 의빠른증식능을이용하여특정 gene 을증폭시킬때 plasmid 이용 (host cell division 시에 plasmid duplication) - origin of replication(ori) sequence 필요 host cell 안에서복제가능하게함 - 복제조절에따라 2 group 으로구분 strigent control ; bacterial cell 분열에조절받음 (1 개씩 replication) relaxed ; bacterial cell 과자율적 (cell 당수백개의 copy 축적 ) pbr322 puc19 puc19 3
GST-Vector (pgex 6p, T7) Eukaryotic expression vector 강원대학교 -유전자조작법 : 생물4년 Isolation of recombinant plasmids 1.E.coli을 EDTA, Glucose 섞인 buffer로현탁 2. SDS, NaOH Mixture 첨가 cell lysis, DNA denature 3. potassium acetate, acetic acid 첨가 neutralization 4. 상층액에 ethanol 이나 isopropanol 첨가 plasmid DNA 침전 5. pellet = clean plasmid DNA 6. 전기영동하여재조합확인 The Boyer-Cohen Cohen- Chang experiment. 1973 Model systems Proof that The Boyer-Cohen Cohen- Chang experiment created a recombinant DNA molecule E. coli - easy and inexpensive to maintain - 사람장내서식, 배설물의주성분 - 해로운박테리아의생장저해 Pathogenic 은 sickness, death 유발 - 돌연변이잘유발 Metabolism 연구에이용 Eukaryote 에서의과정일어나지않음 4
Yeast - Eukaryote와 prokaryote 사이의차이점연구에이용 - Genetic study easy - haploid, single-celled - Grow very rapidly, inexpensive - 2가지종류사용 Saccaromyces cerevisiae; budding Schizosaccaromyces pombe; fission Cloning the yeast origin of replication - yeast genome cut restriction enzyme - fragments clone into plasmid vector - recombinant plasmids growth and select - yeast survive in media lacking leucine - selected yeast cells contain leu2+ gene Nematoda worm - Small and easy to keep - Three days to develop Multicellular organism Caenorhabditis elegans Fruit fly Identification of mutant mutation 특징확인이쉬움 - 짧은시간에많은자손생산 - 알에서성체가되기까지 12일소요 - Drosophila melanogaster Zebrafish - Reproduction rapidly and high number - vertebrate - 발생과정이사람과유사 - 알이투명하여발달확인가능 Danio rerio Amphibians - 알의크기가큼 발생학연구에이용 - 1920년슈페만형성체발견 - Xenopus leavis Chicken 배발생연구에서알사용됨 ; 진화상인간과유사 - 알이크고, 분화관찰에좋음 - Gallus gallus Human cell culture Human blood 나 tissue 에서분리 Primary culture ; derived directly from living tissue - Grow for a short period time and stop - 세포분열횟수제한 Mouse - vertebrate, mammals Human 과더욱가까움 ; 생리학, 발생학적으로유사 - Expensive to maintain, Reproduce slowly Knock out mouse 이용 특정 gene 의기능확인 Plants - Grow slowly, long generation time - 세포벽때문에형질전환이어려움 - Large genome - Transposon 연구에이용 ; corn - Arabidopsis thaliana 5
유전자 cloning Useful Enz. DNA synthesis is done by an enzyme (DNA polymerase) adding nucleotides to the 3 -end of a primer DNA chain Polymerase Chain Reaction (PCR)-1 A pre-defined DNA sequence in the genome can be greatly amplified by repeated Polymerization cycles using 2 primers which hybridize to the ends of the target DNA. In each cycle, the amount of target DNA is doubled. After 10, 20 and 30 cycles, there is a 1000-, million- and billion-fold amplification respectively. Polymerase Chain Reaction (PCR)-2 Each PCR cycle has 3 stepsa. Melting of DNA b. Hybridization of primer c. DNA synthesis 6
Reverse trasncriptase ( 역전사효소 ) 1. 진핵세포로부터 mrna 를분리후 DNA 합성을위한주형으로사용 2. RNA 주형으로부터 DNA 를합성하는것을역전사라한다. 3. DNA단일가닥이만들어진후, RNA가닥은분해된고, 첫번째합성된 DNA가닥을주형으로두번째가닥의 DNA가합성된다. 4. 합성된 DNA에는인트론이존재하지않는DNA (cdna) 로단백질합성에대한유전정보를포함 Restriction endonucleases Restriction endonucleases cut DNA -DNA 의특정 sequence 인지, 절단 (break phosphodiester bond) 발견과정 -1950s; bacteria에서원시적인 immune system 발견 -1960s; enzyme system (E. coli 추출물 ) 발견 self DNA 보호, 외래 DNA 인지- 절단 modification activity (methylation) -1970s; New restriction endonuclease발견 Hind Ⅲ (haemophilus influenzae 에서발견 ) modification activity 없음, 인식자리안의정확한지점절단 Three major class -type Ⅰ, Ⅲ ; restriction and modification activity, 인식자리밖절단, ATP 를에너지원으로사용 예측불가능, ATP 요구성때문에사용안함 -type Ⅱ ; restriction activity 만있음, ATP 필요없음, Mg2+ 필요, 인식자리안이나인접부위예측가능하게절단 DNA 조작에이상적 절단방법 - middle of the site blunt end - 3 of center, 5 of center sticky end Frequency of cutting ; 제한효소가인지하는 sequence의길이에의존 Restriction map ; 제한효소에의해절단한 DNA fragment의크기비교 genetic map과연관 7
Restriction enzymes cleave DNA at a specific sequence Molecular detail of EcoR1 restrictionmodification Cleavage map of the SV40 genome Properties of restriction enzymes-2 HaeIII Haemophilus aegiptius GG/CC Blunt cut Sau3A Staphylococcus aureus /GATC 5 -overhang HhaI Haemophilus haemolyticus GCG/C 3 -overhang SmaI Serratia marcescens CCC / GGG Blunt cut EcoRI Escherichia coli RY13 G / AATTC 5 -overhang PstI Providencia Stuartii CTGCA / G 3 -overhang HaeII Haemophilus aegiptius RGCGC / Y Ambiguous sequence NotI Nocardia otitidis GC / GGCCGC 8 nt sequence 8