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Epicentre Forum 3 (3)
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Ronald Meis, Epicentre Technologies
SequiTherm™ DNA Sequencing Kits were a major advance in sequencing
technology, providing researchers with the advantages of high-temperature
cycle sequencing combined with an enzyme that yields greater accuracy
and longer reads. Nevertheless, some templates contained problematic
regions that were poorly resolved. Incorporating several proprietary
DNA sequencing advancements,* the new SequiTherm
EXCEL Kits have been developed to allow researchers to sequence a broader
range of templates, including many of these difficult templates. In addition,
nonspecific background signal is effectively reduced, resulting in clearer,
less ambiguous data. Sequence data are generated using either standard
cycle sequencing protocols or an alternative high-temperature, isothermal
protocol.
The SequiTherm EXCEL Kits have been optimized to cycle sequence difficult
templates containing regions of high percentage of G+C, inverted repeats,
hairpin structures, and localized areas of interstrand reannealing. The
latter areas are commonly observed with PCR products and certain plasmid
templates. SequiTherm EXCEL Kits can also be used with other extremely
difficult templates (e.g., dinucleotide repeats, homopolymeric stretches)
that give unsatisfactory results with cycle sequencing by using a high-temperature,
isothermal protocol. Here, we demonstrate the advantages of SequiTherm
EXCEL DNA Sequencing Kits for cycle sequencing problematic templates
using manual sequencing with radioactively-labeled primers or automated
sequencing with infrared-fluorescent dye-labeled primers.
Sequencing through secondary structure
pSAD2 is a pUC-based clone containing an insert comprising a 150-base
inverted repeat capable of forming a 75 basepair hairpin/cruciform structure.+ Various
permutations of this structure form at temperatures commonly used in
cycle sequencing reactions. When these structures are encountered by
DNA polymerase, the enzyme is unable to dislodge the basepaired structure
and will pause or terminate primer extension, resulting in 4-lane stops
and extraneous background signal. Figure 1 shows a comparison of DNA
sequence data produced by a standard cycle sequencing kit (lanes A1,
A2) with data produced by the SequiTherm EXCEL Kit (lanes B1, B2). The
reactions were visualized on a LI-COR® 4000 automated DNA Sequencer.
All of the 4-lane stops observed in the standard cycle sequencing reaction
are resolved in the SequiTherm EXCEL reaction. In addition, a reduction
in primer-dimer (arrow) formation by the IRD41-labeled M13 Forward primer
in the SequiTherm EXCELreaction resulted in more readable data in the
lower region of the sequencing ladder.
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Figure 1. SequiTherm EXCEL versus a standard kit using a template
that forms strong secondary structure. pSAD2 DNA was prepared
using an alkaline lysis miniprep procedure, quantified by absorbance
at 260 nm, and cycle sequenced with a standard kit (SequiTherm)
(lanes A1 and A2) and with the SequiTherm EXCEL Kit (lanes B1 and
B2). Sequencing reactions were performed essentially as recommended
by the manufacturer using 100 fmoles of template with 2 pmoles
IRD41-labeled M13 Forward primer. The reaction was performed with
a 3-step cycling profile (30 seconds at 95°C; 30 seconds at
50°C; 1 minute at 70°C), which included an annealing step
that was necessary for primer binding. Lanes A2 and B2 are continuations
of the data shown in lanes A1 and B1, respectively. The double
arrowhead indicates comparable/overlap regions of the gel. The
arrow denotes primer-dimer formation with the IRD41-labeled M13
Forward primer. The horizontal line indicates the axis of symmetry
of the inverted repeat. Data were visualized on a LI-COR Model
4000 DNA Sequencer. All reactions were loaded in the order G A
T C. |
Reducing nonspecific background signal
The SequiTherm EXCEL Kit also decreases premature termination and nonspecific
background in reactions employing radionuclides. Cycle sequencing data
of the pSAD2 template using this detection method is shown in Figure
2. Internal labeling with [alpha-35S]-dATP was used for the
reactions in lanes A1 and A2. Primer end-labeling with [gamma-32P]-dATP
was used for the reactions in lanes B1 and B2. Lanes A1 and B1 show sequence
data obtained using a standard cycle sequencing kit, whereas lanes A2
and B2 show data obtained using the SequiTherm EXCEL Kit. Comparison
of standard reactions shown in Figures 1 (lanes A1, A2) and 2 (lanes
A1, B1) revealed that the number of 4-lane stops and background signal
observed using the pSAD2 template varied with the cycling profile and
detection method used. Nevertheless, the SequiTherm EXCEL Kit resolved
ambiguous regions observed with 35S internal labeling (Figure
2, lane A2), and diminished the background bands observed with 32P
end labeling (Figure 2, lane B2).
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Figure 2. Reduction of nonspecific background using SequiTherm
EXCEL compared with a standard kit. pSAD2 DNA was cycle sequenced
with a standard kit (SequiTherm) (lanes A1 and B1) and with the
SequiTherm EXCEL Kit (lanes A2 and B2). The reaction using 35S
internal label was accomplished using 315 fmoles template with
15 pmoles M13 Forward primer; the reaction with 32P
end-labeled primer was accomplished using 50 fmoles template with
1.5 pmoles 32P-labeled M13 Forward primer. These reactions
were performed using a 2-step cycling profile (30 seconds at 95°C;
1 minute at 70°C), incorporating a combined annealing/extension/termination
step. Internal labeling with 35S was used in lanes A1
and A2. Primer end-labeling with 32P was used in lanes
B1 and B2. Autoradiography was performed for 2 days with 35S
label, and for 1 day with 32P label. All reactions were
loaded in the order G A T C. |
Sequencing through localized areas of strand reannealing
The SequiTherm EXCEL Kit decreases background signal caused when the
polymerase encounters localized areas of reannealing between the template
and complementary DNA strands (Figure 3). Lanes A and B show sequence
data produced from a standard cycle sequencing kit (lane A) or the SequiTherm
EXCEL Kit (lane B) of a globin PCR product. Lanes C and D show sequence
data produced from a standard cycle sequencing kit (lane C) or the SequiTherm
EXCEL Kit (lane D) of pUC19 DNA. In both cases, sequencing reactions
generated by the SequiTherm EXCEL Kit produced less ambiguous data. Of
particular note is the ambiguous base present in Figure 3, lane A (double
arrowhead); the predominant signal appears in the "G" lane. Comparison
of this base call with the corresponding base in lane B shows that the
proper base call is "C."
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Figure 3. SequiTherm EXCEL versus a standard kit using templates
with localized areas of strand reannealing. The 500 bp globin
template was amplified with 30 cycles of PCR and purified by agarose
gel electrophoresis. This purified PCR product was cycle sequenced
with a standard kit (SequiTherm) (lane A) and with the SequiTherm
EXCEL Kit (lane B) using 350 fmoles of template with 2 pmoles of
IRD41-M13 Forward primer. Miniprep pUC19 DNA (100 fmoles of template
with 2 pmoles IRD41-labeled M13 Forward primer) was cycle sequenced
with a standard kit (SequiTherm) (lane C) and with the SequiTherm
EXCEL Kit (lane D). Sequencing the globin PCR product and pUC19
was accomplished as in Figure 1. The double arrowhead indicates
an ambiguous base call. Data were visualized on a LI-COR Model
4000 DNA Sequencer. All reactions were loaded in the order G A
T C. |
In conclusion, the new SequiTherm EXCEL DNA Sequencing Kit offers significant
advantages over standard cycle sequencing for templates that contain
regions of secondary structure (e.g., inverted repeats, hairpins) or
localized reannealing. Moreover, the new kit yields sequence data with
significantly lower background for all DNA templates.
*U.S. and international patents pending
+pSAD2 providec courtesy of Dr. Grant McFadden, Dept. of Biochemistry,
University of Alberta, Edmonton, Alberta, Canada.
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