Different lengths of DNA duplex containing same number of nucleotides were designed in accordance with complementary base pairing principle. Each oligonucleotide pair (50nM, 100μL each) sponsored by IDT Technologies were mixed and placed in the Dynamica C-Master Gradient thermal cycler. Starting at 95ºC, the temperature was then dropped 1ºC per minute until room temperature was reached. Polyacrylamide gel electrophoresis (PAGE) was then conducted to check for the stability of each DNA duplex.
| Oligo | Sequence | Size (nucleotide) |
|---|---|---|
| Oligo 1 | TGCCGGGTATAGCGCTTAGCATCCCGACACCTCTACCTACGCGGCCCAAACCAACAGGGC | 60 |
| Oligo 2 | GCCCTGTTGGTTTGGGCCGCGTAGGTAGAGGTGTCGGGATGCTAAGCGCTATACCCGGCA | 60 |
| Oligo 3 | GGTTCCTCTTGCTTACGGTCGGCGGACTAGGCCGTACCTACCATC | 45 |
| Oligo 4 | GATGGTAGGTACGGCCTAGTCCGCCGACCGTAAGCAAGAGGAACC | 45 |
| Oligo 5 | GCTGTTATGAAATAGCGGGAGGTTAGACGTCAGGT | 35 |
| Oligo 6 | ACCTGACGTCTAACCTCCCGCTATTTCATAACAGC | 35 |
| Oligo7 | TCGTCATAGGCCGCACGAGTTAGAT | 25 |
| Oligo 8 | ATCTAACTCGTGCGGCCTATGACGA | 25 |
| Oligo 9 | TCATCACACTTGGAT | 15 |
| Oligo 10 | ATCCAAGTGTGATGA | 15 |
| Lane | Composition |
|---|---|
| 1 | Oligo 1 |
| 2 | Oligo 2 |
| 3 | Oligo 1 + Oligo 2 |
| 4 | Oligo 3 |
| 5 | Oligo 4 |
| 6 | Oligo 3 + Oligo 4 |
| 7 | Oligo 5 |
| 8 | Oligo 6 |
| 9 | Oligo 5 + Oligo 6 |
| Lane | Composition |
|---|---|
| 1 | Oligo 7 |
| 2 | Oligo 8 |
| 3 | Oligo 7 + Oligo 8 |
| 4 | Oligo 9 |
| 5 | Oligo 10 |
| 6 | Oligo 9 + Oligo 10 |
After selection of the most stable DNA duplex, the sequences were ordered from our sponsor Integrated DNA Technologies. The sequences we ordered were undergone azide modification with an NHS Ester functional group to attach an azide moiety at the 5' of the oligos.
The enzymes, including lignin peroxidase (LiP) and manganese peroxidase (MnP) were engineered using pSBIK3 vector.