Applications
- Automated sequencing of all DNA templates, including those containing
difficult-to-sequence regions that cannot be resolved with other sequencing
kits (Figs. 1-4).
SequiTherm EXCEL II™ Sequencing Kits for automated sequencers use
the SequiTherm EXCEL II Polymerase, which improves sequencing performance
with increased signal intensities and the ability to sequence through GC-rich
regions. The SequiTherm EXCEL II DNA Sequencing Kits incorporate the use
of betaine,* which enables sequencing of a broad range of templates, including
those containing regions that are very difficult to sequence. Protocols are
provided for both cycle and high-temperature isothermal (noncycle) sequencing.
The kits have been optimized for use with the following DNA sequencers:
LI-COR™ 4000 and 4200 series Sequencers, NEN™ Global IR2 DNA
Sequencers, ALF™ DNA Sequencers, and ABI PRISM™ DNA Sequencers
using dye primer technology. Using primers labeled with standard sequencing
dyes, the SequiTherm EXCEL II kits may also be used with ABI PRISM 373 and
377 series sequencers. The kits are not compatible with ABI capillary sequencers
(ABI 310, 3100, or 3700 series) or with BigDye™ technology.
Two different kits are available for use with LI-COR™ and NEN™ sequencing
equipment. The first kit (Cat. No. SE9101LC) contains Termination Mixes that
give optimal read lengths up to about 1,000 bases and is designed for use
on 25- to 41-cm gels. The second kit (Cat. No. SE9202LC) provides enhanced
signal intensities in the resolution range of 1,000-1,400 bases and is ideal
for users interested in obtaining the longest possible reads in conjunction
with 66-cm gels, version 4.x of the BaseImagIR™ software, and all versions
of e-Seq™ software.
Benefits
- Sequence templates containing hairpin structures (Fig. 3), regions of
high GC or AT (Fig. 1) content, areas of interstrand reannealing, or nucleotide
repeats (Fig. 4).
- Reduced background signal results in clearer, less ambiguous sequence
data (Fig. 2).
- Kits compatible with LI-COR, NEN, and ALF DNA sequencers (both dye-primer
and internal label) and with ABI PRISM automated sequencers (dye-primer).
- Optional isothermal protocol to sequence templates that are recalcitrant
to cycle sequencing. Termination Mixes contain 7-deaza-dGTP to reduce band
compression during gel electrophoresis.
Figure 4. Sequencing through highly problematic repeat regions. Supercoiled
plasmids containing GA-dinucleotide repeats (A), GGCtrinucleotide repeats
(B), or GAAA-tetranucleotide repeats (C) were successfully sequenced with
the SequiTherm EXCEL™ II DNA Sequencing Kit using the standard cycle
sequencing protocol and dye-labeled primers.
*Covered by issued and/or pending patents. |
 |
Figure 1. Sequencing of a difficult template. An AT rich template
was sequenced using a standard cycle sequencing kit (A) and the SequiTherm
EXCEL™ II Kit-LC (B). As shown, several strong stops were eliminated
and sequence was easily read using the SequiTherm EXCEL II Kit-LC. Samples
were loaded in the order G,A,T,C. Data were visualized on a LI-COR® Model
4000 DNA Sequencer. |
