PCR mediated mutagenesis 2014년도 2학기 생화학 실험 (2) 6주차 조교 : 장지영, 신진철

Site-specific mutagenesis Introduction. Site-specific mutagenesis 1. Single base mutation. 2. Multiple mutation. 3. Insertion. 4. Deletion. The types of mutagenesis. The cause of mutagenesis. 1. UV. 2. Chemical-Carcinogen. 3. Error prone of PCR. 4. Site-directed mutagenesis.

Overlap Extension PCR 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 뺄 것 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ Primer 2 Primer 1 3’ Primer 3 5’ 3’ 5’ Primer 4 5’ 3’ 5’ 3’ 1 Cycle 1 Cycle 2 Cycle 2 Cycle

Overlap Extension PCR Ligation PCR 1 Cycle 2 Cycle Primer로써 기능!!

Overlap Extension PCR 뺄 것 541 ttcacatgccctcagtgccgaaagagctttcctcggcggagcttc 586 cgccccaacctgcagctggccaatatggtccaggtgattcggcag 631 atgcacccaacccctggtcgagggagccgcgtgaccgatcagggc 676 atctgtcccaaacaccaagaagccctgaagctcttctgcgaggta 721 gacgaagaggccatctgtgtggtgtgccgagaatccaggagccac 766 aaacagcacagcgtggtgccattggaggaggtggtgcaggagtac 811 aaggccaaactgcaggggcacgtggaaccactgaggaagcacctg 856 gaggcagtgcagaagatgaaagccaaggaggagaggcgagtgaca 901 gaactgaagagccagatgaagtcagagctggcagcggtggcctcg 946 gagtttgggcgactgacacggtttctggctgaagagcaggcaggg

Experimental Procedure : Gel Extraction 1. Agarose gel : membrane binding solution = 10 mg : 10 ㎕ 씩 넣어 55℃ heat block에서 10분간 녹인다. 2. 잘 녹았는지를 vortexing 을 통해 확인 후, sample을 column으로 옮긴다. 3. 14000rpm, 1min centrifuge 4. Wash buffer 750 ㎕ 를 넣은 후 14000rpm, 1 min centrifug 5. 한번 더 14000rpm, 1 min centrifuge하여 남은 wash buffer를 제거한다. 6. Column을 새 tube에 옮긴 후 D.W.를 30 ㎕ 를 넣고 5분을 기다린다. 7. 14000rpm, 5 min centrifuge

Experimental Procedure : Ligation PCR PCR condition Ligation PCR PCR condition Component Quantity per reaction Targets 1-10kb Distilled water 34 ㎕ 5x Herculase II reaction buffer 10 ㎕ dNTP mix 0.5 ㎕ DNA template(1+3) 1 ㎕ DNA template(2+4) Primer 1+4 2.5 ㎕ Herculase II fusion DNA polymerase Total reaction volume 50 ㎕ Temp.(℃) Time 95 2 min 30 sec 55 72 1 min 5 min 30 cycles

PCR fragment for cloning into T-vector T-vector cloning. PCR fragment for cloning into T-vector PCR polymerase 의 종류 Taq DNA polymerase - 3’→5’ exonuclease 활성 없음 특이적으로 3’말단에 A가 생김. Pfu DNA polymerase 3’→5’ exonuclease 활성 있음 3’ 말단에 A가 생기지 않음 < alkaline phosphatase, Calf intestinal(CIP) >

Lac Z 을 이용한 selection Inhibitor Lactose Galactose Lactose β-galactosidase + Glucose X-Gal Blue color β-galactosidase + Glucose X-gal used to indicate whether a bacterium expresses the β-galactosidase enzyme, which is encoded by the lac Z gene, in a technique called blue/white screening.

Lac Z 을 이용한 selection

Amp/LacZ를 이용한 selection

PCR construct (insert) Experimental Procedure 1. T-vector ligation Ingredient Volume ( ul) 2x ligation buffer 5 T-vector 0.5 PCR construct (insert) 3.5 T4 DNA ligase 1 Total 10 Ligation time 1. Overnight at 16 ℃ 2. 1 ~ 2 hours at Room temperature ◈ Ligation volume setting ng of vector ng of insert : = 1 : 5 size of vector size of insert

Experimental Procedure 2. Transformation Positive control Self-ligation No insert Control insert (542bp)

Experimental Procedure 3. Bacteria culture 16hr 4. Plasmid DNA Extraction 5. Cloning confirmation using two-cut digestion

Membrane Wash Solution Membrane Binding Solution Report – 결과 및 고찰 1. Gel extraction solution 의 원리 조사 2. Lac operon원리와 selection의 원리에 대해서 쓰시오.(원리) 3. 수업자료에 있는 PCR의 2번 째 cycle 에서 합성되는 DNA를 그려보세요. 4. PCR-mediated PCR primer design (Red blank 인 부분을 deletion하는 construct, primer 4개 design 할 것) Design한 primer의 Tm 값과 annealing temperature를 구하시오. (Tm-5℃) Membrane Wash Solution 10mM potassium acetate (pH 5.0) 2. 80% ethanol 3. 16.7μM EDTA (pH 8.0) Membrane Binding Solution 1. 4.5M guanidine isothiocyanate 2. 0.5M potassium acetate

Report - 결과 및 고찰 1 atggctgccgttgccatgacacccaaccctgtgcagacccttcag 46 gaggaggcggtgtgcgccatctgcctcgattacttcacggacccc 91 gtgtccatcggctgcgggcacaacttctgccgagtttgtgtaacc 136 cagttgtggggtggggaggatgaggaggacagagatgagttagat 181 cgggaggaggaggaggaggacggagaggaggaggaagtggaggct 226 gtgggggctggcgcggggtgggacacccccatgcgggatgaagac 271 tacgagggtgacatggaggaggaggtcgaggaggaagaagagggt 316 gtgttctggaccagtggcatgagcaggtccagctgggacaacatg 361 gactatgtgtgggaggaggaggacgaggaggaagacctggactac 406 tacttgggggacatggaggaggaggacctgaggggggaggatgag 451 gaggacgaggaggaagtgctggaggaggttgaggaagaggatcta 496 gaccccgtcaccccactgcccccgcctccagcccctcggaggtgc 541 ttcacatgccctcagtgccgaaagagctttcctcggcggagcttc 586 cgccccaacctgcagctggccaatatggtccaggtgattcggcag 631 atgcacccaacccctggtcgagggagccgcgtgaccgatcagggc 676 atctgtcccaaacaccaagaagccctgaagctcttctgcgaggta 721 gacgaagaggccatctgtgtggtgtgccgagaatccaggagccac 766 aaacagcacagcgtggtgccattggaggaggtggtgcaggagtac 811 aaggccaaactgcaggggcacgtggaaccactgaggaagcacctg 856 gaggcagtgcagaagatgaaagccaaggaggagaggcgagtgaca 901 gaactgaagagccagatgaagtcagagctggcagcggtggcctcg 946 gagtttgggcgactgacacggtttctggctgaagagcaggcaggg 991 ctggaacggcgtctcagagagatgcatgaagcccagctggggcgt 1036 gcgggagccgcggctagtcgccttgcagaacaggccgcccagctc 1081 agccgcctgctggcagaggcccaggagcggagccagcaggggggt 1126 ctccggctgctccaggacatcaaggagactttcaataggtgtgaa

Report 제출기한 : 2014-10-23 오전까지(~12시) (늦게 제출 하면 감점) Hand-writing 제출기한 : 2014-10-23 오전까지(~12시) (늦게 제출 하면 감점) Hand-writing 미 제출 시 0점 처리 조교: 장지영(010-9493-9222) jyjang0809@naver.com