Cleavage of deoxyuracil close to the ends of dsDNA and ssDNA
| dsDNA | ssDNA | |||
|---|---|---|---|---|
| Enzyme | Base pairs from the 5' End | Base pairs from the 3' End | Nucleotides from the 5' End | Nucleotides from the 3' End |
| USER® Enzyme (M5505) | 3 | 6 | 9 | 6 |
| Thermolabile USER II Enzyme (M5508) | 3 | 3 | 12 | 6 |
| Thermostable USER III Enzyme (M5509) | 3 | 3 | 12 | 3 |
| Thermostable Endonuclease Q (M0701) | 6 | 3 | 6 | 3 |
As a general rule, the modified base should be at least 3 base pairs from either end of the dsDNA for efficient cleavage. The modified base should be at least 6-12 nucleotides from the 5’ end and 3-6 nucleotides from the 3’ end of ssDNA for efficient cleavage.
Method: Annealed 5´ FAM labeled double-stranded or single-stranded DNA oligos, designed to have a deoxyuracil the indicated number of base pairs from either the 5’ or 3’ end of the DNA, were incubated with the indicated enzyme (1 unit/ 1pmol oligo) for 60 minutes at the recommended incubation temperature and Reaction Buffer. Cleaved fragments were resolved and analyzed by capillary electrophoresis.
