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

2. 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.

3. 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

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

5. 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

6. 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.
rpm, 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.
rpm, 5 min centrifuge

7. 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

8. 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) >

9. 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.

10. Lac Z 을 이용한 selection

11. Amp/LacZ를 이용한 selection

12. 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

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

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

15. 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
μM EDTA (pH 8.0)
Membrane Binding Solution
1. 4.5M guanidine isothiocyanate
2. 0.5M potassium acetate

16. 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

17. Report 제출기한 : 2014-10-23 오전까지(~12시) (늦게 제출 하면 감점) Hand-writing
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