Molecular analysis of TB-Bead extracts
A. Introduction
It is clear that many of our customers are using TB-Beads to extract mycobacteria from
sputum and then use the extracted mycobacteria to perform molecular analysis. Used in this
way, TB-Beads can minimise or even eliminate centrifugation during sample preparation
which:
simplifies the extraction
reduces risk from infectious aerosols
enables high-throughput of samples with the potential to lead to an automated
extraction protocol
To date, the TB-Bead extraction has been reported to be compatible with several
amplification systems including commercial PCR systems provided by Cepheid, Hain, and
Roche. In addition, the TB-Bead extraction has been reported to be compatible with
isothermal amplification methods such as the RNA amplification method provided by Tosoh
and the LAMP method provided by the Eiken Chemical Company.
Using the TB-Bead extraction protocol a concentrated suspension of mycobacteria is
derived from sputum. Before molecular analysis can be performed, the nucleic acids have to
be extracted from these mycobacteria. To do this the mycobacteria have to be ruptured to
release the nucleic acids and then these nucleic acids can be used directly or after
purification. There are a number of different methods available:
Incubation in chaotrope such as 5 M guanidinium
thiocyanate Heating in the presence of strong alkali and
detergent Bead-beating
Sonication
Some of these approaches are available as commercial products and have been shown to
work well. In our laboratory we have used the first three approaches and the simple
protocols are given below.
B. TB-Bead extraction of mycobacteria for molecular analysis
The standard TB-Bead extraction method should be used. Following elution of the TB from
the beads as described in the protocol, take 250µl of the bead-free eluate and centrifuge in
a microfuge (small centrifuge) for 5 min at 12,000 rpm. After centrifugation, remove the
liquid being careful not to disturb the pellet. Follow one of the protocols below:
C. Nucleic Acid Extraction
i) Chaotrope release of DNA
1. To the pellet from B above, add 25 µl of 0.05 M NaOH, 0.1% (v/v) Tween20 and heat
at 95ºC for 10 min.
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Molecular analysis of TB-Bead extracts
2. The nucleic acids can now be prepared using Qiagen nucleic acid purification kits
such as the MinElute PCR Purification kit, Catalogue Number 28004. Simply follow
the kit protocol by adding sequentially 200µl DNA Lysis Buffer, 500µl DNA Binding
Buffer and 20µl DNA Beads, as described in the Qiagen protocol. Follow the Qiagen
protocol from this point.
ii) Heating in the presence of strong alkali and detergent to release the DNA
1. Resuspend the pellet from B above in 250 µl of water and recentrifuge as before.
Remove the liquid being careful not to disturb any pellet.
2. Add 50 µl of 0.05 M NaOH, 0.1% (v/v) Tween20 to the pellet and heat at 95ºC for 10
min.
3. Add 50µl of 0.05 M HCl, 50 mM Tris pH 7.5 to neutralise.
4. Use for PCR analysis.
iii) Release of the DNA by Bead beating
1. Resuspend the pellet from B above in 250 µl of water and recentrifuge as before.
Remove the liquid being careful not to disturb any pellet.
2. To the pellet, add 50 µl 10 mM Tris, 1 mM EDTA, 0.1% (v/v) Triton X-100, 0.1% (v/v)
Tween20.
3. Using a pipette tip with the tip cut-off, add 100µl of a 80% (w/v) slurry of glass beads
(<106 µm, acid-washed glass beads, Sigma, Catalogue number G4649) in water.
4. Place the tubes in a vortex genie 2 and bead-bead at maximum speed for 10 min.
5. Use the supernatant (the liquid above the settled beads) for PCR analysis.
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