uDumBell – Circularization of rv0678 for genotypic bedaquiline resistance testing of Mycobacterium tuberculosis

Jason D Limberis

Published: 2023-02-28 DOI: 10.17504/protocols.io.36wgqj9zyvk5/v1

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

1.
AB
ComponentVolume (ul)
5X Reaction Buffer10
5X Q5 High GC Enhancer10
10 mM dNTPs1
Forward primer2.5
Reverse primer2.5
DNA (5ng)2
Q5 High-Fidelity DNA Polymerase1.5
Nuclease-Free Water20.5

PCR using primer set

ABCD
StepTemp (C)Time (s)Cycles
Denaturation98301
Denaturation981034
Annealing6210
Extension7220
Extension7221

Cycle parameters

Adapter ligation

2.

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)

3.
AB
ComponentVolume (ul)
T4 DNA Ligase Buffer (10X)2
PCR product (upto 1ug), as low as 50ng, probably much lower possible)10
dumbell adapter3
Ligase (add last, don’t vortex)1
H204

Incubate as below, with the lid temperature set to 40°C

AB
TempMinutes
2230
15120
4120
655
4.

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

5.
AB
ComponentVolume (ul)
NEBuffer 4 (10x)1
Exonuclease VIII (truncated)1
DNA18

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

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