0  1  2  3  4  5  6  0

1. 3 4 3 1 7 3 0 11 11 start & end 3 3 6 5 3 3 9 3 2 5 0  1  2  3  4  5  6  0

2. Step 1. Hybridization • 올리고머 개수 : 7 + 24 + 5 = 36개 • 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

3. Step 2. Ligation • 효소 : T4 DNA ligase (TaKaRa) • 반응액 구성 올리고머 용액 50 ml 10X ligation buffer 10 ml ligase solution 2 ml (700 Unit) ddH2O to to 100 ml • 반응 온도 : 16℃ • 반응 시간 : O/N

4. O0 O1 O2 O3 O4 O5 O6 O0 O0  1 O1  2 O2  3 O3  4 O4  5 O5  6 O6  0

5. O0 Step 3. PCR I • 와 로 PCR 100 pmol of each primer to a total volume of 60 ml processed for 35 cycles at 94℃for 15 s/ at 30℃for 60 s O0 cycle denaturation (95℃) annealing (53℃) polymerization (72℃) 1 5 min 1 min 1 min 2~35 1 min 1 min 1 min 36 1 min 1 min 5 min

6. Step 4. Gel Electrophoresis I • 반응에 참여하지 않은 dNTP 등을 제거하기 위해서 전기영동

7. Oi O4 Step 5. Affinity Chromatography Biotin Sterptavidin magnetic particle 5’ ssDNA 0 1 2 3 4 5 6 0 Biotin Sterptavidin 0 1 2 3 4 5 6 0

8. 1. O_0 and biotinylated ^O_0 를 primer로 사용하여 PCR  ssDNA 2. Aaffinity purification with ^O_2, ^O_3, ^O_4, and ^O_5 3. PCR amplification during affinity purification process

9. Obtaining ssDNA 1. PCR 증폭 : O_0와 biotinylated ^O_0 를 사용하여PCR 증폭 2. Annealing to streptavidin paramagnetic particles : Incubating in 100 ml of 0.5X saline sodium citrate (SSC) for 45 min at RT with constant shaking 3. Washing: washed three times in 200 ml of 0.5X SSC 4. Denaturation to ssDNA : Heated to 80℃ in 100 ml of ddH2O for 5 min to denature the bound dsDNA 5. Acquisition of ssDNA :The aqueous phase with single-stranded DNA was retained

10. Aaffinity purification 1. Annealing of probe : 1 nmol of biotinylated ^O_i was annealed to particles 2. Washing Wash three times in 400 ml of 0.5  SSC for 45 min at RT with constant shaking 3. Removal of unbound ssDNA Particles were washed four times in 400 ml of 0.5  SSC to remove unbound ssDNA and then 4. Denaturation to ssDNA 5. Acquisition of ssDNA

11. Step 6. Gel Electrophoresis/ PCR • Affinity purification에 의해 얻은 solution을 전기영동한다. • 그 중에서 가장 작은 분자량을 가지는 밴드를 잘라서 elution한다. • 다시 PCR과 전기영동 과정을 반복하면서 purity를 높인다.

12. Step 7. Sequencing • PCR product를 sequencing한다. • 예상한 sequence와 동일한지 확인한다.

13. vertex_0 fragment_3 vertex_1 fragment_3 vertex_2 ATGGGGCTTT GTTGCGTCTT GAGTGGAGAG GTGTCACGTC GCTACCGGAA CACTTAGGTG CCGCGGCGCCCCGCCGC CCGCGGCGCCCCGCCGC CAACGCAGAA GGCGCCGCGGGGCGGCG CTCACCTCTC CACAGTGCAG GGCGCCGCGGGGCGGCG CGATGGCCTT edge_01 edge_12

14. 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