uDumBell – Circularization of rv0678 for genotypic bedaquiline resistance testing of Mycobacterium tuberculosis
Jason D Limberis
Abstract
The ligation of dumbbell (hairpin) oligos to linear dsDNA produces pseudo-circular DNA. Including deoxyUridine in the PCR primer sequences causes Q5 and other high-fidelity polymerases to arrest elongation. This results in overhangs that were successfully ligated to a complementary hairpin structure. The deoxyUridine reduced the PCR product by approximately two-thirds, but this was ameliorated by increasing the Q5 DNA polymerase concentration three-fold.
Attachments
Steps
Amplicon PCR
| A | B |
|---|---|
| Component | Volume (ul) |
| 5X Reaction Buffer | 10 |
| 5X Q5 High GC Enhancer | 10 |
| 10 mM dNTPs | 1 |
| Forward primer | 2.5 |
| Reverse primer | 2.5 |
| DNA (5ng) | 2 |
| Q5 High-Fidelity DNA Polymerase | 1.5 |
| Nuclease-Free Water | 20.5 |
PCR using primer set
| A | B | C | D |
|---|---|---|---|
| Step | Temp (C) | Time (s) | Cycles |
| Denaturation | 98 | 30 | 1 |
| Denaturation | 98 | 10 | 34 |
| Annealing | 62 | 10 | |
| Extension | 72 | 20 | |
| Extension | 72 | 2 | 1 |
Cycle parameters
Adapter ligation
Prepare the dumbell (hairpin) by incubating at 80°C followed by cooling to room temperature over 0h 30m 0s(this only needs to be done once)
| A | B |
|---|---|
| Component | Volume (ul) |
| T4 DNA Ligase Buffer (10X) | 2 |
| PCR product (upto 1ug), as low as 50ng, probably much lower possible) | 10 |
| dumbell adapter | 3 |
| Ligase (add last, don’t vortex) | 1 |
| H20 | 4 |
Incubate as below, with the lid temperature set to 40°C
| A | B |
|---|---|
| Temp | Minutes |
| 22 | 30 |
| 15 | 120 |
| 4 | 120 |
| 65 | 5 |
Incubate at Room temperature for 0h 5m 0s
Place on amagnetic rack
Aspirate supernatant
Add 200µL 70% (v/v) ethanol
Wait for 0h 0m 30s
Aspirate and discard the supernatant
Add 200µL 70% (v/v) ethanol
Wait for 0h 0m 30s
Aspirate and discard the supernatant
Resuspend beads in 20µL of H20
Incubate for 0h 2m 0s
Transfer to a clean PCR tube
Exonuclease treatment – optional
| A | B |
|---|---|
| Component | Volume (ul) |
| NEBuffer 4 (10x) | 1 |
| Exonuclease VIII (truncated) | 1 |
| DNA | 18 |
Incubate at 37°C for 0h 30m 0s
Stop reaction by adding EDTA to at least 11 mM.
Heat Inactivation 70°CC for 0h 30m 0s
