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Epicentre Forum 4 (2)
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Ronald Meis, Epicentre Technologies
The SequiTherm EXCEL™ DNA Sequencing Kits are particularly useful
for difficult-to-sequence DNA templates. Examples of such templates include
those containing inverted repeat regions (hairpins), short tandem repeat
regions (homopolymeric, di-, tri-, and tetranucleotide repeats), and
those capable of forming localized areas of interstrand reannealing such
as long tandem repeats, highly AT-rich templates, highly GC-rich templates,
and PCR products. While the new technology incorporated into the SequiTherm
EXCEL products achieved this result, it also required the use of increased
amounts of DNA template compared to the standard SequiTherm™ Cycle
Sequencing Kit. Here, we demonstrate that the new SequiTherm EXCEL II
DNA Polymerase, recently incorporated into the SequiTherm EXCEL Kits,
has greatly improved sensitivity for low amounts of template. This increased
sensitivity not only enables the use of less template than that recommended
for the original SequiTherm EXCEL DNA Polymerase (SequiTherm EXCEL I
DNA Polymerase), but also comparable or lower amounts of template than
are recommended for SequiTherm DNA Polymerase. In addition, the
increased sensitivity of SequiTherm EXCEL II DNA Polymerase permits shorter
autoradiographic exposure times. It has also allowed the development
of simple, high-temperature isothermal sequencing protocols applicable
to users of both radioactive and LI-COR® automated DNA sequencing
systems.
Increased sensitivity using a 32P-end-labeled primer
The data shown in Figure 1 demonstrate several of the advantages of
SequiTherm EXCEL II DNA Polymerase when sequencing a highly GC-rich template.
A plasmid clone containing the CGG-trinucleotide repeat portion of a
normal human FMR1 (Fragile-X associated) gene was sequenced using a 32P-5'-end
labeled primer in a cycle sequencing reaction with either SequiTherm
EXCEL I DNA Polymerase or SequiTherm EXCEL II DNA Polymerase. Reaction
1 was performed with SequiTherm EXCEL II DNA Polymerase and 80 fmoles
of template; Reaction 2 was performed with SequiTherm EXCEL I DNA Polymerase
and the same amount of template. Unlike a standard cycle sequencing reaction
(data not shown), both reactions were able to resolve the CGG-trinucleotide
repeat region of the clone. Clearly, the intensity of the sequencing
ladder produced by SequiTherm EXCEL II DNA Polymerase was much greater
than that produced by SequiTherm EXCEL I DNA Polymerase when equal amounts
of template were used. The data shown in Figure 1 were visualized after
only a 3-hour autoradiographic exposure. Nearly 100% of the ladder in
Reaction 1 was clearly readable after a 1-hour exposure (data not shown).
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Figure 1. Comparison of SequiTherm EXCEL I and II DNA Polymerases
using an end-labeled primer. M13 Forward Primer (Epicentre)
was 5-end labeled with 32P and cycle sequencing was performed according
to the SequiTherm EXCEL DNA Sequencing Kit instructions using 80
fmoles of a supercoiled template containing the (CGG)23 repeat
region of the human FMR1 gene. Cycling parameters were: 95°C
for 5 minutes, followed by 30 cycles of 95°C for 30 seconds,
70°C for 1 minute. Three microliters of the final reaction
mixture were loaded into each well of an 8% polyacrylamide, 7M
urea gel in 1X glycerol tolerant buffer. The gel was fixed for
20minutes in 20% ethanol, transferred to blotting paper, dried,
and subjected to autoradiography on X-Omat XAR-351 film (Kodak)
for 3 hours. |
Increased sensitivity using 35S internal labeling
Similar comparative results were also achieved using an [alpha-35S]-dATP
internal-labeling protocol. Supercoiled pUC19 DNA was cycle sequenced
using 100, 200, or 500 fmoles of template with either SequiTherm EXCEL
I or SequiTherm EXCEL II DNA Polymerase. The results are shown in Figure
2. Reactions 1 and 3 were performed with SequiTherm EXCEL II DNA Polymerase,
whereas Reactions 2, 4, and 5 were performed with SequiTherm EXCEL I
DNA Polymerase. SequiTherm EXCEL II DNA Polymerase clearly produced greater
signal intensities with both 200 fmoles (Reaction 1 vs. 2) and 100 fmoles
(Reaction 3 vs. 4) of template. The intensity of the signal produced
using 100 fmoles of template and SequiTherm EXCEL II DNA Polymerase (Reaction
3) was also greater than that produced with 500 fmoles of template (the
amount recommended for many standard cycle sequencing kits) and SequiTherm
EXCEL I DNA Polymerase (Reaction 5). The data shown in Figure 2 were
visualized after a 4-hour autoradiographic exposure, and nearly 80% of
the ladder in Reaction 1 was clearly readable after a 1-hour exposure
(data not shown).
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Figure 2. Comparison of SequiTherm EXCEL I and II DNA Polymerases
using an 35S-internal-labeling protocol. Supercoiled
pUC19 was cycle sequenced using M13 Forward Primer (Epicentre)
and [alpha-35S]-dATP according to the SequiTherm EXCEL
DNA Sequencing Kit instructions. Cycling and gel parameters were
as described for Figure 1 except autoradiographic exposure time
was 4 hours. |
Use of SequiTherm EXCEL II DNA Polymerase in automated sequencing
reactions visualized on a LI-COR sequencer
Automated DNA sequencing reactions are also improved using SequiTherm
EXCEL II DNA Polymerase. To test this, pSAD2, a difficult-to-sequence
template containing a 75 bp hairpin structure, was used as a template
in cycle sequencing reactions using a LI-COR IRD41-labeled M13 Forward
primer. The data were visualized on a LI-COR Model 4000 Automated DNA
Sequencer (Figure 3). Sixty-five fmoles of supercoiled template were
sequenced using either SequiTherm EXCEL II DNA Polymerase (Reaction 1)
or SequiTherm EXCEL I DNA Polymerase (Reaction 2). The band intensity
difference between the two reactions is substantial. Additionally, the
ladder produced by SequiTherm EXCEL II DNA Polymerase with 65 fmoles
of pSAD2 (Reaction 1) is not only darker than that produced by SequiTherm
EXCEL I DNA Polymerase when 4-fold more template (260 fmoles) is used
(Reaction 3), but it is also darker than the ladder produced by SequiTherm
EXCEL I DNA Polymerase with 65 fmoles of nicked circular pSAD2 (Reaction
4). Similarly, much higher signal intensities were achieved by cycle
sequencing with SequiTherm EXCEL II DNA Polymerase using an easy-to-sequence
template as shown in Figure 4.
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Figure 3. Improved signal intensities using SequiTherm EXCEL
II DNA Polymerase with a difficult-to-sequence template read on
a LI-COR automated sequencer. Supercoiled pSAD2 was cycle sequenced
using IRD41-labeled M13 Forward primer according to the SequiTherm
EXCEL Long-Read™ DNA Sequencing Kit- LC instructions. Cycling
parameters were: 95°C for 5 minutes, followed by 30 cycles
of 95°C for 30 seconds, 50°C for 15 seconds, 70°C for
1 minute. One microliter of the final reaction mixture was loaded
into each well (in the order G, A, T, C) of a 6% Long-Ranger™ polyacrylamide
(FMC), 7M urea gel in 1X TBE Buffer, and visualized on a LI-COR
Model 4000 DNA Sequencer. |
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Figure 4. Improved signal intensities using SequiTherm EXCEL
II DNA Polymerase with supercoiled pUC19 read on a LI-COR automated
sequencer. Primer, cycling, and gel parameters were
as described in Figure 3. |
Use of SequiTherm EXCEL II DNA Polymerase with a high-temperature
isothermal protocol
The increased signal intensities with SequiTherm EXCEL II DNA Polymerase
has also allowed development of a simple high-temperature isothermal
(single-cycle) sequencing protocol for use with the LI-COR Model 4000
Sequencer (Figure 5). There are several advantages of this protocol.
The first is faster "reaction-to-gel" times. This is due to: 1) the shorter
time required to perform a single cycle rather than the normal 30 cycles
of a cycling reaction; and 2) eliminating the acid-nicking step for supercoiled
templates that is required with SequiTherm EXCEL I DNA Polymerase and
the alkaline denaturation step necessary with modified T7 DNA polymerase.
Only thermal denaturation is necessary. The second advantage of the isothermal
sequencing protocol is less ambiguous data on difficult-to-sequence templates.
Whereas cycle sequencing with the SequiTherm EXCEL II system works on
a vast array of difficult templates, we have encountered some templates
that are recalcitrant to cycle sequencing. In our hands, a high-temperature
isothermal reaction with SequiTherm EXCEL II DNA Polymerase was the only
way to resolve many of these templates.
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Figure 5. High-temperature isothermal sequencing using SequiTherm
EXCEL II DNA Polymerase and visualized on a LI-COR automated sequencer. A
high-temperature isothermal procedure was used to sequence 566
fmoles of supercoiled pUC19. Isothermal reaction parameters were:
95°C for 10 minutes, cool to 40°C over 5 minutes, 65°C
for 5 minutes. Primer and gel parameters were as described in Figure
3. |
We have demonstrated several of the advantages of the new SequiTherm
EXCEL II DNA Polymerase over SequiTherm EXCEL I DNA Polymerase. These
advantages include: 1) increased signal intensities for 32P, 35S,
and IRD41 dye-primer detection systems; 2) the use of less template for
cycle sequencing reactions; 3) decreased autoradiographic exposure times
in many instances; and 4) the ability to use dsDNA templates in a high-temperature
isothermal protocol using only thermal denaturation. SequiTherm EXCEL
II DNA Polymerase is now included in all SequiTherm EXCEL and SequiTherm
EXCEL Long-Read DNA Sequencing Kits.
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