1

0

3

2 5

6

4

0 1 2 3 4 5 6 0

start&end

3

5

3

3

7

11

3

3

9

11

3

3

Step 1. Hybridization

올리고머 개수 : 7 + 24 + 5 = 36 개 올리고머 양 : 50 pmol

Tm : vertex : 50~60℃

edge : 60~95℃

fragment (weight) : -65~80 ℃

초기온도 100℃ 점차적으로 온도를 낮추어 37℃ 상온까지 내린다 .

0.5℃/0.5 min

Step 2. Ligation

효소 : T4 DNA ligase (TaKaRa) 반응액 구성

올리고머 용액 50 l

10X ligation buffer 10 l

ligase solution 2 l (700 Unit)

ddH2O to to 100 l

반응 온도 : 16℃ 반응 시간 : O/N

O0 O1 O2 O3 O4 O5 O6

O2 3 O3 4O1 2O0 1 O4 5 O5 6

O0

O6 0

Step 3. PCR I

와 로 PCR100 pmol of each primer

to a total volume of 60 l

processed for 35 cycles at 94℃for 15 s/ at 30℃for 60 s

O0 O0

cycle

1

2~35

36

denaturation (95 )℃ annealing (53 )℃ polymerization (72 )℃

5 min

1 min

1 min

1 min

1 min

1 min

1 min

1 min

5 min

Step 4. Gel Electrophoresis I

반응에 참여하지 않은 dNTP 등을 제거하기 위해서 전기영동

Step 5. Affinity Chromatography

BiotinSterptavidin

5’ssDNA

Oi

0 1 2 3 4 5 6

BiotinSterptavidin

O4

magneticparticle

0

0 1 2 3 4 5 6 0

1. O_0 and biotinylated ^O_0 를 primer 로 사용하여 PCR ssDNA

2. Aaffinity purification with ^O_2, ^O_3, ^O_4, and ^O_5

3. PCR amplification during affinity purification process

Obtaining ssDNA

1. PCR 증폭: O_0 와 biotinylated ^O_0 를 사용하여 PCR 증폭

2. Annealing to streptavidin paramagnetic particles: Incubating in 100 l of 0.5X saline sodium citrate

(SSC) for 45 min at RT with constant shaking

3. Washing : washed three times in 200 l of 0.5X SSC

4. Denaturation to ssDNA : Heated to 80℃ in 100 l of ddH2O for 5 min to

denature the bound dsDNA

5. Acquisition of ssDNA:The aqueous phase with single-stranded DNA was

retained

Aaffinity purification

1. Annealing of probe: 1 nmol of biotinylated ^O_i was annealed to

particles

2. WashingWash three times in 400 l of 0.5 SSC for 45

min at RT with constant shaking

3. Removal of unbound ssDNAParticles were washed four times in 400 l of 0.5

SSC to remove unbound ssDNA and then

4. Denaturation to ssDNA 5. Acquisition of ssDNA

Step 6. Gel Electrophoresis/ PCR

Affinity purification 에 의해 얻은 solution 을 전기영동한다 .

그 중에서 가장 작은 분자량을 가지는 밴드를 잘라서 elution 한다 .

다시 PCR 과 전기영동 과정을 반복하면서 purity 를 높인다 .

Step 7. Sequencing

PCR product 를 sequencing 한다 . 예상한 sequence 와 동일한지 확인한다 .

GAGTGGAGAG GTGTCACGTCATGGGGCTTT GTTGCGTCTT GCTACCGGAA CACTTAGGTG

CAACGCAGAA GGCGCCGCGGGGCGGCG CTCACCTCTC

CCGCGGCGCCCCGCCGC

vertex_0 fragment_3 vertex_1

edge_01

CCGCGGCGCCCCGCCGCfragment_3

CACAGTGCAG GGCGCCGCGGGGCGGCG CGATGGCCTT

edge_12

vertex_2

Discussion

Negative PCR result Sequence Design

: high GC content possibility in non-specific binding Hybridization

: temperature decrease: non-specific binding nick formation no ligation rxn

Ligation: temperature (16℃ vs RT) & incubation time (O/N vs 4

hr): enzyme unit

Solutions Ligation/Hybridization conditions Substrate addition order