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Epicentre Forum 5 (4)
The BESS MutaScan™, BESS T-Scan™, and BESS G-Tracker™ Mutation
Detection and Localization Kits offer simple, rapid scanning methods
to locate and identify point mutations, frameshifts, deletions, insertions,
and repeat expansions in PCR-amplified DNA. Here, we present answers
to the most frequently asked questions about the BESS kits.
What does the BESS (base excision sequence scanning) procedure entail?
The BESS procedure involves a single PCR amplification step using a
fluorescent-, radioactive-, or biotin-labeled primer in the prescence
of a limiting concentration of dUTP. The amplification product is then
used in two separate reactions to identify T and G changes. In the T-specific
reaction, the uracil-containing PCR product is treated with Uracil N-Glycosylase
and Endonuclease IV. This reaction produces a nested set of fragments
that are easily separated on a sequencing gel. In the G-specific reaction,
the PCR product is first treated with the BESS G-Tracker Modification
Reagent in the presence of visible light. The modified G is removed by
the BESS G-Tracker Excision Enzyme Mix, generating nested fragments that
are characterized in a manner similar to the T reaction. The T and G
reactions produce results analogous to T and G sequencing ladders. The
results may be analyzed using manual sequencing gels or automated DNA
sequencers. Reagents for both T and G scanning reactions are supplied
in the BESS MutaScan™ Kit. T and G scanning reagents are available
separately in the BESS T-Scan™ Kit and BESS G-Tracker™ Kit,
respectively.
What are the most important parameters for successful BESS reactions?
Generation of a single amplification product in sufficient quantity
is important to the success of the reaction. PCR primer design is important.
If the primers are designed properly, there should be minimal primer-dimer
formation, a single amplification product should result, and yields should
be sufficient. Primers should be 30-40 bases from the target sequence
to be analyzed for optimal results. In addition, a suitable detection
method should be used.
What size fragment can be analyzed with the BESS system?
PCR products of 100-700 base pairs are optimal, but larger products
may be analyzed with radioactively labeled primers and equipment that
can produce the read lengths required.
What kinds of non-radioactive detection can be used?
For fluorescent detection, primers labeled with TET and FAM dyes generate
stronger signal intensities than HEX. Cy3, Cy5, IRD700, and IRD800 dyes
are not compatible with BESS G-Tracker reactions. Chemiluminescent detection
using a number of different systems (e.g., PhotoTope® Detection Kit,
New England BioLabs) has been performed in conjunction with a biotinylated
PCR primer.
How does BESS compare to T- and G-lane sequencing?
BESS reactions produce accurate results analogous to T and G sequence
ladders, but the method is faster than sequencing. In BESS, PCR products
are used directly to produce nested fragments in less time than it takes
to perform cycle sequencing of PCR products. Sequencing can then be used
to confirm the mutation results.
Can I detect all point mutations using the BESS system?
By analyzing both strands with BESS G-Tracker and BESS T-Scan reactions,
it is possible to determine 100% of all mutations in the fragment analyzed.
Can I analyze both strands of the PCR product simultaneously?
Yes, by using two primers in the PCR reaction, each carrying a different
fluorescent label for detection.
Does the PCR product require purification before performing the BESS
reactions?
No, if the PCR product is a single product at sufficient concentration.
My amplification reaction containing dUTP generates additional PCR
fragments, whereas my normal amplification reaction produces a single
PCR product. Why would this happen?
A number of things could be the cause of this problem. First, the template
concentration may be too high. For a single-copy gene, use approximately
103-104 copies of a mammalian template (e.g., 10-100
ng), 104-105 copies of a bacterial template (0.1-1
ng), and proportionally less for organisms with smaller genomes. Second,
if the additional fragments are of low molecular weight, then primer-dimer
formation may be the problem. In this instance, lowering the concentration
of primers in the amplification reaction (or redesigning the primers)
and performing a hot start may help. Another consideration is the choice
of thermostable DNA polymerase used. In our experience, Taq DNA polymerase
incorporates dUTP efficiently into PCR products; whereas other polymerases
may not synthesize dUTP-containing PCR products as efficiently.
Can I use a Taq/proofreader mix in my amplification reaction?
While a Taq/proofreading enzyme mixture will work in BESS reactions,
it is not necessary. Any particular misincorporated base is present at
too low of a frequency in the PCR product mixture to be detected. In
addition, proofreading polymerases alone do not incorporate dUTP efficiently
and therefore, we do not recommend their use.
What type of light box is required for the BESS G-Tracker reactions?
The BESS G-Tracker reactions require a light box with about 5,000 lux,
like those used for viewing autoradiographic films.
| BESS MutaScan™ Mutation Characterization Kit |
BESS-T&G™ Base Reader Kit |
| BESS T-Scan™ Mutation Detection and Localization Kit |
BESS-T™ Base Reader Kit |
| BESS G-Tracker™ Mutation Detection and Localization Kit |
BESS-G™ Base Reader Kit |
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