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°C
C for 0h 30m 0s