DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBace DNA Analysis Systems - Product Booklet GE Healthcare
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GE Healthcare
DYEnamic ET
Dye Terminator Cycle
Sequencing Kit for
MegaBace DNA
Analysis Systems
Product Booklet
Codes: US81090
US81095Page finder
1. Legal 3
2. Handling 6
2.1. Safety warnings and precautions 6
2.2. Quality control 6
2.3. Storage 6
3. Components of the kit 7
4. Materials not supplied 9
5. Introduction 10
6. Important considerations for using this kit 12
7. Protocols 14
7.1. Preparation of sequencing reactions 15
7.2. Post-reaction cleanup 18
7.3. Resuspension of samples 20
7.4. Instrument setup and data analysis 21
7.5. Injection and run parameters 22
8. Appendixes 23
8.1. Appendix 1: Template DNA—general considerations 23
8.2. Appendix 2: Primers—general considerations 26
8.3. Appendix 3: Cycling conditions 27
8.4. Appendix 4: Considerations for post-reaction cleanup 29
9. Troubleshooting 31
10. References 36
21. Legal
GE and GE monogram are trademarks of General Electric Company.
AutoSeq, DYEnamic, MegaBACE, Sephadex, Sequenase, TempliPhi
and Thermo Sequenase are trademarks of GE Healthcare
companies.
This kit is sold pursuant to Authorization from PE Applied Biosystems
under one or more of the following U.S. Patents: 4,849,513;
4,855,255; 5,015,733; 5,118,800; 5,118,802; 5,161,507; 5,171,534;
5,242,796; 5,306,618; 5,332,666; and 5,366,860, and corresponding
foreign patents and patent applications. The purchase of this kit
includes limited non-transferable rights (without the right to resell,
repackage, or further sublicense) under such patent rights to use
this kit for DNA sequencing or fragment analysis, solely when used
in conjunction with an automated instrument for DNA sequencing
or analysis which have been authorized for such use by Applied
Biosystems, or for manual sequencing. Purchase of this product
does not itself convey to the purchaser a complete license or right
to perform automated DNA sequence and fragment analysis under
the subject patents. No other license is hereby granted for use of
this kit in any other automated sequence analysis instrument. The
rights granted hereunder are solely for research and other used that
are not unlawful. No other license is granted expressly, impliedly, or
by estoppel.
Further information on purchasing licenses to perform DNA
sequence and fragment analysis may be obtained by contacting the
Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive,
Foster City, California 94404.
GE HEALTHCARE IS LICENSED AS A VENDOR FOR AUTHORIZED
SEQUENCING AND FRAGMENT ANALYSIS INSTRUMENTS.
3NOTICE TO PURCHASER ABOUT LIMITED LICENSE
The purchase of this kit (reagent) includes a limited non-exclusive
sublicense under certain patents* to use the kit (reagent) to perform
one or more patented DNA sequencing methods in those patents
solely for use with Thermo Sequenase II DNA polymerase purchased
from GE Healthcare for research activities. No other license is granted
expressly, impliedly or by estoppel. For information concerning avail-
ability of additional licenses to practice the patented methodologies,
contact GE Healthcare Bio-Sciences Corp, Director, Business
Development, 800 Centennial Avenue, PO Box 1327, Piscataway, NJ
08855 USA.
* US Patent numbers 4,962,020, 5,173,411, 5,409,811, 5,498,523,
5,614,365 and 5,674,716.
Patents pending.
† This product is sold under licensing arrangements with Roche
Molecular Systems, F Hoffmann-La Roche Ltd and the Perkin-
Elmer Corporation. Purchase of this product is accompanied by
a limited license to use it in the Polymerase Chain Reaction (PCR)
process for research in conjunction with a thermal cycler whose
use in the automated performance of the PCR process is covered
by the up-front license fee, either by payment to Perkin-Elmer or as
purchased, i.e. an authorized thermal cycler.
Energy Transfer dyes and primers—US Patent numbers: 5,654,419,
5,688,648, and 5,707,804.
T7 Sequenase DNA polymerase—This reagent (kit) is covered by or
suitable for use under one or more US Patent numbers: 4,795,699;
4,946,786; 4,942,130; 4,962,020; 4,994,372; 5,145,776; 5,173,411;
5,266,466, 5,409,811, 5,498,523 and 5,639,608. Patents pending in US
and other countries.
Thermo Sequenase II DNA polymerase—Patent pending.
© 2006 General Electric Company – All rights reserved.
4GE Healthcare reserves the right, subject to any regulatory and
contractual approval, if required, to make changes in specification
and features shown herein, or discontinue the product described at
any time without notice or obligation.
Contact your GE Healthcare representative for the most current
information and a copy of the terms and conditions.
http//www.gehealthcare.com/lifesciences
GE Healthcare UK Limited.
Amersham Place, Little Chalfont,
Buckinghamshire, HP7 9NA UK
52. Handling
2.1. Safety warnings Warning: This kit contains
formamide. This protocol also
and precautions
requires the use of ethanol, a
Warning: For research use
flammable liquid. Gel reagents
only. Not recommended
may contain acrylamide, a
or intended for diagnosis
neurotoxin and suspected
of disease in humans or
carcinogen. Please follow
animals. Do not use internally
the manufacturer’s Material
or externally in humans or
Safety Data Sheet regarding
animals.
safe handling and use of these
All chemicals should be materials.
considered as potentially
hazardous. We therefore 2.2. Quality control
recommend that this product is All batches of DYEnamic ET Dye
handled only by those persons Terminator Cycle Sequencing
who have been trained in Kit for MegaBACE are assayed
laboratory techniques and according to the recommended
that it is used in accordance starting point protocol
with the principles of good described in this booklet.
laboratory practice. Wear The reactions are analyzed
suitable protective clothing on MegaBACE sequencing
such as laboratory overalls, instrument. Specifications
safety glasses and gloves. for release are based on
Care should be taken to avoid assessment of sequence by
contact with skin or eyes. In length of read (> 500 bases),
the case of contact with skin accuracy and signal quality.
or eyes wash immediately
with water. See material safety
2.3. Storage
data sheet(s) and/or safety
Store at -15°C to -30°C
statement(s) for specific advice.
63. Components of the kit
Solutions included in DYEnamic™ ET Dye Terminator Cycle
Sequencing Kit for MegaBACE™ DNA Analysis Systems have been
carefully formulated for optimal sequencing results. Each reagent
has been tested extensively and its concentration adjusted to meet
GE Healthcare standards. It is strongly recommended that reagents
supplied in the kit be used as described in this protocol.
The following components are included in the kit:
Kit component US81090 US81095
(500 rxns) (10 000 rxns)
DYEnamic ET 4 x 1 ml 1 x 80 ml
terminator
reagent premix
(MegaBACE)
Ammonium 1 x 1 ml 1 x 20 ml
acetate (7.5 M
ammonium
acetate)
Control 1 x 200 μl Not included
M13mp18
DNA
(single-stranded,
0.2 μg/μl)
Control primer 1 × 400 μl Not included
(universal
cycle primer
5’-GTTTTCCCAGTCACGACGTTGTA-3’)
(2.0 pmol/μl)
Loading solution 1 x 10 ml 1 x 200 ml
(70% formamide,
1 mM EDTA)
7Store these kits and their components at -15°C to -30°C (NOT in a
frost-free freezer). When the reagents are not in a freezer, keep them
on ice prior to use. For convenience, the kit can be stored at 2–4°C
for up to three months with no loss of performance; however, this
should be avoided if the reagents will not be completely consumed
within three months.
World Wide Web address
http//www.gehealthcare.com/lifesciences
Visit the GE Healthcare home page for regularly updated product
information.
84. Materials not supplied
Reagents
• Water—Use only deionized, distilled water for the sequencing
reactions.
• Sequencing primers—Use primers appropriate for the template
being sequenced. For most applications, 5 pmol of primer is
sufficient.
• Ethanol (95–100% and 70%)—For post-reaction cleanup.
Note: Do NOT use denatured alcohol.
• Electrophoresis matrix for MegaBACE—Long-read Matrix
(US79676) for capillary electrophoresis. This is linear
polyacrylamide (LPA) matrix.
Equipment
• Liquid-handling supplies—Vials, pipettes, centrifuge and vacuum
centrifuge. All sequencing reactions should be run in plastic
microcentrifuge tubes (typically 0.5 ml) or 96-well or 384-well
plates suitable for thermal cycling.
• Instrument—This kit is used with MegaBACE sequencing
instrument.
• Thermal cycler—For thermally cycled incubations between 50°C
and 95°C (1–100 cycles).
95. Introduction
DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBACE
DNA Analysis Systems is designed for sensitive and robust
sequencing with MegaBACE sequencing system. Exploiting the
capabilities of the instrument, the superior resolving properties of
LPA long-read matrix, the sensitivity of DYEnamic ET terminators, and
the robust performance of Thermo Sequenase™ II DNA polymerase,
the kit provides a convenient and flexible dye terminator format for
high throughput sequencing and industry-leading data quality.
To use this product, a sequencing reaction premix is combined
with template DNA and primer and thermally cycled. The reaction
products are then precipitated with ethanol or isopropanol to
remove unincorporated dye-labelled terminators. Samples are finally
dissolved in a loading solution for separation and detection using
MegaBACE sequencing instrument.
Energy transfer dye terminator-based sequencing
DYEnamic ET Terminator Kits are based on a modification of
traditional dideoxynucleotide chain termination chemistry (1) in
which terminators are labeled with fluorescent dyes for automated
detection. In this case, however, each of the four dideoxy
terminators—ddG, ddA, ddT and ddC—is labeled with two dyes—
fluorescein and one of four different rhodamine dyes—rather than a
single dye (2, 3). Fluorescein has a large extinction coefficient at the
wavelength (488 nm) of the argon ion laser used in the sequencing
instrument. Acting as the donor dye, fluorescein absorbs energy
from incident laser light and transfers it to the rhodamine acceptor
dye on the same terminator molecule. Each acceptor dye then emits
light at its characteristic wavelength for detection, identifying the
nucleotide that terminated extension of the DNA fragment. This
energy transfer format (4) is more efficient than direct excitation of
10the acceptor dye by the laser, and produces a sequencing method
that is very sensitive and robust.
The acceptor dyes used in the kits are the same standard rhodamine
dyes—rhodamine 110, rhodamine-6-G, tetramethyl rhodamine, and
rhodamine X—used in earlier Taq dye terminator methodologies,
so the DYEnamic ET reaction products can be detected on any
instrument that can monitor the original Taq dye terminator
chemistry.
The kit also features dITP, as well as Thermo Sequenase II DNA
polymerase, a thermostable enzyme that efficiently incorporates
dITP. By replacing dGTP with dITP, even very strong compression
artifacts common to high GC-content DNA are resolved for more
accurate data interpretation.
Thermo Sequenase II DNA polymerase
Thermo Sequenase II DNA polymerase is a thermostable DNA
polymerase specifically engineered for cycle sequencing by
GE Healthcare. The enzyme readily accepts dideoxynucleotide
terminators (5) and generates bands of uniform intensity, much like
T7 Sequenase™ DNA polymerase (6, 7). Its tolerance to high salt
conditions, efficient utilization of dITP, high processivity, and excellent
performance on GC-rich templates make it an efficient and robust
sequencing enzyme.
Cycle sequencing
Thermostable DNA polymerases allow sequencing reactions to be
cycled through alternating periods of thermal denaturation, primer
annealing, and extension/termination to increase the signal levels
generated from template DNA (8–13). This amplification process
employs a single primer, so the amount of product increases linearly
with the number of cycles. A cycling protocol is especially useful
when the amount of template is limiting or the sensitivity of the
detection system is low.
116. Important considerations for using
this kit
The reagent formulations and DNA polymerase used in this product
differ from those in other sequencing kits. This DYEnamic ET Dye
Terminator Kit (US81090 and US81095) is specifically designed to
provide the longest reads and the greatest degree of success using
MegaBACE sequencing systems. For optimal results, the following
parameters should be noted:
1. For each 20 μl reaction volume, 8 μl of premix should be used.
This ratio MUST be maintained for optimal results. If using a 384-
well format, use 4 μl of premix for each 10 μl reaction volume. No
other configuration is supported.
2. The DYEnamic ET terminator dye set is compatible with the
standard MegaBACE sequencing filters and beam splitters.
Table 1. MegaBACE filter and beam splitter assignments
Filters Beam splitters
MegaBACE 500 520DF20, 555DF20, 540DRLP and 595DRLP
and 1000 585DF20, and 610LP
MegaBACE 4000 520DF20, 555DF20, 570DRXR, 540DRLP
585DF20, and 610LP and 595DRLP
3. The ethanol/salt and isopropanol precipitation protocol
recommended for post-sequencing cleanup has been carefully
developed to provide an efficient and low-cost method and should
be followed exactly as described for optimal results.
4. The metal ions in the enzyme reaction mix are optimized for the
enzymes included in the premix. Therefore, template DNA and
primer should be resuspended in either water (preferably) or in a
buffer containing no more than 0.1 mM EDTA. If nmol quantities of
Mg2+, EDTA or other metal ion chelators are introduced with
12template or primer, increased failure rates, weak signals or short
read-lengths may occur.
5. Prolonged denaturation steps (> 1 minute at 95°C) should be
avoided during the cycling protocol since enzyme denaturation is
likely with weak signals and failed reactions resulting.
6. Extension times < 4 minutes and extension temperatures > 60°C
can be used with Thermo Sequenase II DNA polymerase. One
minute at 60°C is suggested for extension.
137. Protocols
Preliminary preparations and general handling instructions
Thaw and maintain all kit reagents on ice prior to use. Whenever
possible, cap the tubes to minimize evaporation of the small
volumes of reagents used. Dispense reagents using disposable-tip
micropipettes, and exercise caution to avoid contamination of stock
solutions. Thoroughly mix reaction mixtures after each addition
by “pumping” the solution two or three times with a micropipettor
without creating air bubbles. Centrifuge briefly tubes/plates to
collect the reaction mixtures at the bottoms of the vessels. With
practice, reactions can be completed in 15–20 minutes.
The protocol described below provides high-quality sequencing
results using the control DNA and primer provided in the kit.
However, this protocol should be regarded only as a starting point.
Optimization of protocols might be necessary to obtain the best
sequencing results for specific templates. Please refer to Appendixes
1–4 and the trouble-shooting section for additional information to
help optimize the sequencing reactions.
147.1. Preparation of sequencing reactions
Researchers who utilize 0.5 ml tubes should follow the sequencing
reaction and post-reaction cleanup instructions specified for
96-well plates.
1. Assemble each sequencing reaction as follows:
96-well format
Template DNA __ μl
Primer __ μl
Water __ μl
Sequencing reagent premix 8 μl
Total volume 20 μl
Note: Adjust the amount of distilled water such that the total
volume of DNA, primer and water is 12 μl. When combined with
8 μl of sequencing reagent premix, the total volume of the reaction
mix should be 20 μl.
384-well format
Template DNA __ μl
Primer __ μl
Water __ μl
Sequencing reagent premix 4 μl
Total volume 10 μl
Note: Adjust the amount of distilled water such that the total
volume of DNA, primer and water is 6 μl. When combined with 4 μl of
sequencing reagent premix, the total volume of the reaction mix
should be 10 μl.
15Note: 0.1–1 μg (40–400 fmol) of single-stranded DNA or 0.2–2 μg
(80–800 fmol) of double-stranded plasmid DNA and 5 pmol of
primer are recommended for routine sequencing. The volumes
of DNA and primer added to each reaction will depend on their
concentrations. Dilute the DNA and the primer in deionized water
or buffer containing no more than 0.1 mM EDTA. Do not use buffers
containing > 0.1 mM EDTA since they may reduce the effective metal
cofactor concentration in the reactions. For additional information
concerning the amount of template and primer to use in the
reaction, see Appendixes 1 and 2 on pages 21–25.
Note: The most consistent results are obtained when sequencing
reagent premix is used at full strength. No other configuration is
supported.
2. Assemble the control reaction exactly as follows:
96-well format
M13mp18 control template 1 μl
Control primer 2.5 μl
Water 8.5 μl
Sequencing reagent premix 8 μl
Total volume 20 μl
384-well format
M13mp18 control template 1 μl
Control primer 2.5 μl
Water 2.5 μl
Sequencing reagent premix 4 μl
Total volume 10 μl
16Note: The sole purpose of the control reaction is to confirm the
performance of the sequencing premix under specified and tested
conditions. It is crucial to assemble and perform the reactions
exactly as described above. Customer data can then be compared
with GE Healthcare quality control data if the performance of the
sequencing premix is in doubt.
3. After dispensing all reagents, cap the tubes or seal the plates.
Mix thoroughly by gentle vortexing or gentle pumping (to avoid
bubbles) with a pipettor. Centrifuge briefly to bring contents to the
bottom of the tubes or wells.
4. Place the tubes or plate into the thermal cycler. Run the
following cycling program for 25 cycles:
95°C, 20 seconds
50°C, 15 seconds
60°C, 1 minute
(Cycling is complete in about 1 hour)
Note: For additional information concerning cycling conditions, see
Appendix 3 on page 25.
5. After cycling is complete, centrifuge the tubes/plate briefly to
collect the reaction mixtures at the bottoms of the tubes/wells.
177.2. Post-reaction cleanup
Please refer to Appendix 4 for additional information.
1. Option 1—Ethanol precipitation
1.1. Add 2 μl (96-well plate) or 1 μl (384-well plate) of
7.5 M ammonium acetate to each reaction tube or well.
1.2. Add 55 μl (96-well plate) or 27.5 μl (384-well plate) of 100%
ethanol or 60 μl (96-well plate) or 30 μl (384-well plate) of
95% ethanol to each reaction and mix by inverting the plate
several times (do not vortex). The final concentration of ethanol
should be 70%. It is not necessary to use cold ethanol nor is
it necessary to incubate the samples at low temperature for
precipitation.
Note: This step is critical. Final ethanol concentrations < 65%
produce weak signals while concentrations > 75% result
in sequences with “blob” artifacts due to precipitation of
unincorporated dye terminators.
1.3. Centrifuge tubes at either room temperature or 4°C in a
microcentrifuge for 15 minutes at ~12 000 rpm. Centrifuge 96-
well or 384-well plates for at least 30 minutes at 2 500 x g or
greater.
1.4. Remove the supernatant from each microcentrifuge tube by
aspiration. For plates, a brief inverted spin (1 minute at 300 x g)
is sufficient for supernatant removal. Remove as much liquid as
possible at this step to prevent dye blobs.
1.5. Wash the DNA pellets with 70% ethanol. Use as large a volume
of 70% ethanol as the tube or well can accommodate safely.
Centrifuge briefly.
Note: Scientists at GE Healthcare routinely use 250–500 μl for 0.5-ml
microcentrifuge tubes, 100 μl for 96-well plates, and 45 μl for 384-
well plates.
181.6. Remove the supernatants by aspiration or by an inverted spin.
Air-dry (preferably) or vacuum-dry (in a vacuum centrifuge) the
pellets for 2–5 minutes. Do NOT overdry.
2. Option 2—Isopropanol Precipitation
2.1. Add 40 μl (96-well plate) or 20 μl (384-well plate) of 80%
isopropanol to each reaction and mix well using a vortex mixer.
Note: Good results are obtained using a final concentration of
40–65% isopropanol in the precipitation mix; 50–60% isopropanol is
optimal. It is important to utilize an isopropanol solution that is less
than 100% isopropanol because the addition of pure isopropanol,
even to the same final concentration, produces dye blobs. These
blobs are caused by very high local concentrations of isopropanol
before and during mixing. Please see Appendix 4 for further details.
2.2. Centrifuge tubes at either room temperature or 4°C in a
microcentrifuge for 15 minutes at ~12 000 rpm. Centrifuge 96-
well or 384-well plates for at least 30 minutes at 2 500 x g or
greater.
2.3. Remove the supernatant from each microcentrifuge tube by
aspiration. For plates, a brief inverted spin (1 minute at 300 x g)
is sufficient for supernatant removal. Remove as much liquid as
possible at this step to prevent dye blobs.
Note: DNA pelleted by isopropanol precipitation is less firm than DNA
isolated after ethanol precipitation, and can be lost during wash and
inverted spins at high relative centrifugal force.
2.4. Wash the DNA pellets with 70% ethanol; DNA precipitated by
isopropanol should also be washed with 70% ethanol. Use
as large a volume of 70% ethanol as the tube or well can
accommodate safely. Centrifuge briefly.
Note: Scientists at GE Healthcare routinely use 250–500 μl for 0.5–ml
microcentrifuge tubes, 100 μl for 96-well plates, and 45 μl for 384-
well plates.
192.5. Remove the supernatants by aspiration or by an inverted spin.
Air-dry (preferably) or vacuum-dry (in a vacuum centrifuge) the
pellets for 2–5 minutes. Do NOT overdry.
3. Alternatives to ethanol and isopropanol precipitation include the
GE Healthcare AutoSeq™96 product line (See Appendix 4). For
more information, contact your local GE Healthcare office or visit
us at http//www.gehealthcare.com/lifesciences and search with
the keyword, “Autoseq96”.
7.3. Resuspension of samples
1. Dissolve each pellet in 10 μl of MegaBACE loading solution
and vortex vigorously for 10–20 seconds to ensure complete
resuspension. Briefly centrifuge to collect the samples at the
bottom of the well and to remove bubbles.
General recommendations
a) The DNA pellet MUST be completely dissolved at this step for
optimal sequencing results. If a fixed angle rotor was used for
centrifugation, the DNA pellet will be on the side of the well. This
material must be washed to the bottom of the well to ensure
that the entire reaction product is injected onto the MegaBACE.
b) It is not necessary to heat samples prior to injection. Heating
samples can cause excessive evaporation of the resuspension
buffer and speed the breakdown of the dye-labeled sequencing
products.
207.4. Instrument setup and data analysis
1. For instrument setup and data analysis, please refer to the
instrument documentation supplied with the MegaBACE
sequencing instruments.
2. Under the Plate Setup tab of the MegaBACE Instrument Control
Manager (ICM), select New to create new instrument parameters.
3. Set up instrument parameters as follows:
MegaBACE 500 and 1000
Matrix Fill / High Pressure Time: 200 seconds
Matrix Fill / Relaxation Time: 20 minutes
Prerun: 5 minutes
Prerun Voltage: 9 kV
Matrix Flush Time 1: 20 seconds
Matrix Flush Time 2: 7 seconds
Low-Pressure Time: 240 seconds
User Input Time: 120 seconds
Preinjection Time: 15 seconds
MegaBACE 4000
Matrix Fill / High Pressure time: 120 seconds
Matrix Fill / Relaxation Time: 1 minute
Prerun: 5 minutes
Prerun Voltage: 9kV
Matrix Flush Time: 20 seconds
Low-Pressure Time 1: 5 seconds
Low-Pressure Time 2: 240 seconds
User Input Time: 120 seconds
Preinjection Time: 15 seconds
4. Proceed with the New Plate procedure as described in the
instrument documentation.
217.5. Injection and run parameters
1. For maximum reproducibility, use a voltage < 5 kV for injection
(2 or 3 kV is standard). Injection time can be varied widely during
optimization with injections as short as 5 seconds or as long as
400 seconds being equally successful. A recommended starting
point for injection from MegaBACE loading solution is 2 kV for
75 seconds. For optimal results, the product of the time and
voltage of injection should be within the range of 100 to 200
kV seconds. If spin columns or gel filtration plates are used, it is
convenient to leave samples in the eluent and directly inject. In
this case, a recommended starting point is 3 kV for 75 seconds
with optimal results obtained between 150 and 270 kV seconds.
These parameters are suggested starting points that are suitable
for the control template in the kit and for most samples. If
signals are low, longer injection times might prove beneficial. If
sample overloading is a problem, utilize shorter injection times as
discussed within the troubleshooting section.
2. Using standard run conditions of 100 minutes at 9 kV, average
read-lengths > 500 bases with 98.5% accuracy can be expected
with the control template. To take advantage of the superior
resolving power of the LPA matrix and to achieve the longest
read-lengths, electrophoresis should occur for 200 minutes at
6 kV. Even with these recommendations, the quality and quantity
of the template remain the most important factors affecting read
length and success rate.
228. Appendixes
8.1. Appendix 1: Template DNA—general
considerations
Template amount
This protocol typically produces optimal results using 100–200 fmol
of template DNA, but these numbers should be considered as
guidelines. In some cases, more or less template can be used due
to the sensitivity and robustness of DYEnamic ET terminators. For
example, scientists at GE Healthcare have obtained good results
with the MegaBACE sequencing systems using 25–500 ng
(10 fmol–200 fmol) of pure, (single-stranded) M13mp18 DNA. For
routine sequencing, follow the guidelines described above.
The following formula calculates the optimal mass (0.15 pmol) of
double-stranded template to include in a sequencing reaction:
Mass of template (ng) = Total length of DNA (in base pairs) x 0.1
For example, plasmid that is 3800 base pairs in total length (vector
plus insert) should produce optimal data using ~ 380 ng in these
protocols. The recommended range for template amount is
250–500 ng (100–200 fmol).
These relationships are shown graphically in the Figures 1 and 2. The
best sequencing results are obtained using quantities of template
that fall within the ranges indicated by the dashed lines.
23Short PCR products
140
120
Mass of DNA (in ng)
100
80
60
40
20
0
0 200 400 600 800 1000
Length of template (base pairs)
Fig 1. Recommended mass of template DNA in sequencing reactions
(PCR products)
Plasmids and large PCR products
1400
1200
1000
Mass of DNA (in ng)
800
600
400
200
0
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10 000
Length of template (base pairs)
Fig 2. Recommended mass of template DNA in sequencing reactions
(plasmids, large PCR products)
24Insufficient template DNA present in the sequencing reaction can
produce low signal strengths (< 1 000) that can cause poor base-
calling and short reads. In contrast, too much template can overload
the capillaries or yield very high signal strengths (> 10 000), causing
software miscalls. Excessive template DNA can also deplete the
supply of nucleotides in the sequencing premix and lead to short
sequence reads. This is especially problematic with Polymerase
Chain Reaction (PCR) products where small mass amounts of DNA
are required to provide the optimal picomole amount of template.
Recommended buffer for dilution of DNA template
Dilute the DNA template in water (preferably) or in a weakly buffered
solution containing no more than 0.1 mM EDTA. A suitable buffer is
10 mM Tris-HCl (pH 8.5), 0.1 mM EDTA. This concentration of EDTA is
lower than in typical TE buffers because excess EDTA in the template
or primer resuspension buffer can inhibit sequencing reactions by
reducing the effective magnesium concentration.
Preparation of template DNA
Template of suitable quality for use with DYEnamic ET terminator
kits can be prepared using a variety of procedures and commercially
available products.
Single-stranded plasmid DNA
Several published methods are available for preparing single-
stranded DNA from clones in M13 vectors and hybrid plasmid-phage
(phagemid) vectors (14, 15).
Preparation of double-stranded plasmid DNA
Sequencing double-stranded templates with this product requires no
changes in the protocol—for example, alkaline denaturation is not
required. For optimal results, use plasmid DNA purified by cesium
chloride gradients, polyethylene glycol (PEG) precipitation, adsorption
to glass, columns, and other common DNA purification methods.
25Due to the very small quantity of template used in the reactions,
even less-pure DNA samples can yield acceptable sequence data.
Although there are many popular protocols for purifying plasmid
DNA from 2 ml to 10 ml cultures, GE Healthcare scientists achieve
consistent success using boiling (16, 17) and alkaline (18) mini-prep
methods.
TempliPhi™
Templates for sequencing can also be prepared using TempliPhi
DNA sequencing template amplification kits manufactured by
GE Healthcare (for more details contact your local GE Healthcare
office or visit us at http//www.gehealthcare.com/lifesciences
and search with the keyword, “TempliPhi”). A variety of templates
can be amplified by rolling circle amplification using Phi29 DNA
polymerase and sequencing quality DNA can be prepared within
4–6 hours directly from bacterial colonies (19). Microgram quantities
of template DNA can be prepared at isothermal conditions from
picogram amounts of starting material. Amplified DNA can be used
directly for cycle sequencing without purification.
8.2. Appendix 2: Primers—general
considerations
Primer amount
The optimal amount of primer for sequencing with these protocols is
5 pmol. If too little primer is used, signals may be weak. If too
much primer is present, non-specific priming can occur, resulting
in “noisy” sequences characterized by high background or double
(superimposed) sequences. Excessive primer also can contribute
to an artifact known as the “cliff effect” that typically appears as
50–200 bases of strong peaks in the beginning of the sequence
abruptly followed by weak peaks. The likely cause of this artifact is
the inadvertent generation of PCR products during cycle sequencing
26which accumulate rapidly and deplete the nucleotide supply in the
sequencing premix.
Determine the concentration of your primer and include 5 pmol in
each sequencing reaction (2 pmol for the control primer supplied in
this kit). The concentration of the primer can be measured by the
following method:
Resuspend the primer in water (preferably) or in buffer containing no
more than 0.1 mM EDTA, and determine its optical density at
260 nm (OD260). For primers containing N bases (measured in a
cuvette with a 1 cm path length), the approximate concentration
(pmol/μl) is given by the formula:
Concentration (pmol/μl) = OD260/(0.01 x N)
where N is the number of bases.
Designing a sequencing primer
The length and sequence of a primer determines its melting
temperature and specificity. For cycling temperatures recommended
in this protocol, the primer should be ~ 18–25 bases in length.
The sequence of the primer should be checked for potential self-
annealing or hairpin formation, especially at its 3’-end. Possible sites
of false priming in the vector or other known sequences should also
be identified, again stressing matches involving the 3’-end of the
primer.
8.3. Appendix 3: Cycling conditions
Of the three steps that comprise the cycling program (denaturation,
annealing, and extension), denaturation is the most critical. While
Thermo Sequenase II DNA polymerase has significant advantages
over other DNA polymerases used for cycle sequencing, it is not
as stable against thermal inactivation. The reaction buffer in the
sequencing premix has been specially formulated to protect the
stability of the enzyme and, with proper precautions, Thermo
27Sequenase II DNA polymerase has ample stability for robust
sequencing.
Denaturation step
Important! Do NOT use a denaturation temperature > 95°C or
longer than 30 seconds. A long denaturation step prior to cycling
is commonly employed in PCR, but is unnecessary and not
recommended for cycle sequencing reactions. Extended denaturing
can prematurely inactivate Thermo Sequenase II DNA polymerase
and ultimately produce weak signals.
Annealing step
The appropriate annealing temperature varies with the length and
sequence of the primer. In general, temperatures from 45 to 55°C
are appropriate. An annealing step is usually required only with
primers < 20 bases in length. Optimal annealing temperatures are up
to 5°C higher in DYEnamic ET terminator reactions than with other
dye terminator sequencing products. For primers with sufficiently
high melting temperatures, the annealing step can be omitted, and
a two-step cycling program, alternating between denaturation and
extension temperatures, can be used.
Extension step
Extension at 60°C for 60 seconds is optimal. Thermo Sequenase II
DNA polymerase incorporates dITP more rapidly than other DNA
sequencing enzymes, hence there is no apparent advantage to
increase the time or temperature of the extension step.
Number of cycles
Twenty five to thirty cycles are sufficient to sequence the
recommended amounts of plasmids or PCR products. More cycles
are usually not necessary and may lead to artifacts. Increasing the
number of cycles might be appropriate when sequencing extremely
large templates such as bacterial artificial chromosomes (BACs).
288.4. Appendix 4: Considerations for
post-reaction cleanup
This appendix provides a summary of the considerations for post-
reaction cleanup. These recommendations are starting points for
optimization—the duration of precipitation, length and speed of
centrifugation, geometry of centrifuge rotor, and other parameters
might need adjusting.
AutoSeq96 plates
Unincorporated dye terminator is efficiently removed using
AutoSeq96 filtration plates (27-5340-10). AutoSeq96 is a 96-well
spin plate containing prehydrated G-50 Sephadex™. Follow the
instructions that accompany the plates. The purified sequencing
product is recovered in approximately 20 μl of water.
384-well plates and reduced volume reactions
Many researchers choose smaller reaction volumes for sequencing
reactions performed in a 384-well microplate. In this case, the
standard ethanol and isopropanol precipitation protocols can
be scaled to match the desired reaction volume. For instance,
if the total reaction volume is half of the recommended volume
(5 μl instead of 10 μl), use half of the recommended volume
of ammonium acetate (0.5 μl instead of 1 μl) and half of the
recommended volume of 95% ethanol (15 μl instead of 30 μl).
Success rates might be unacceptable with such small reactions.
Alternately, isopropanol-mediated precipitation can be used.
Isopropanol precipitation in 384-well plates
Isopropanol has two advantages over ethanol: 1) Lower
concentrations are required for precipitation, hence smaller total
volumes are involved during cleanup and 2) It is unnecessary
to add salt to the reaction. After cycling, add 1.5–2.5 volumes of
80% isopropanol. As discussed in detail in protocol step 2.3, the
29disadvantage with isopropanol precipitation is that the pelleted
DNA is more prone to loss during washings and inverted spins. An
isopropanol solution that is less than 100% isopropanol must be
used to avoid forming dye blobs, as explained in protocol step 2.1.
309. Troubleshooting
Prior to diagnosing problems associated with the sequencing
reaction chemistry, operation of the MegaBACE instrument
should be verified for optimal performance by injecting a plate of
MegaBACE M13 DNA Sequencing Standards (US79678) and carrying
out electrophoresis according to the accompanying protocol. If the
average overall read-length of this standard plate is < 500 bases
(98.5% accuracy), routine instrument maintenance, such as capillary
cleaning or focusing, might be required. For further details, contact
GE Healthcare Technical Service for assistance.
Note: Control reagents in the kit should always be run in parallel
with test samples during optimization.
Problem: Sequencing signals are weak.
Weak signals with capillary sequencing can be difficult to
troubleshoot, especially for researchers accustomed to slab gel
sequencing. Weak signals can be the result of an unsuccessful
sequencing reaction, or the inefficient injection or overinjection of
reaction products.
Possible causes/solutions
1. The ionic strength of the loading solution was too high. Electro-
kinetic injection into capillaries is more efficient if the ionic
strength of the loading solution is low.
2. Samples were overloaded. Overloaded samples frequently have
low signals since the peaks are broad and diffuse, and it is
common to misdiagnose overloading as insufficient signal. Under
optimal conditions, detection of all samples should begin within
a few minutes of each other. Samples with late starts and broad
peaks are overloaded. With capillary sequencing, signal may often
be improved by injecting less sample rather than more.
313. The injection conditions were not optimal. Confirm that the
recommended injection conditions were used. Change the
injection conditions by reducing and increasing the duration of
injection three-fold.
4. Too much ethanol was used for precipitation. Excess ethanol will
precipitate salts, buffers and contaminants in the template DNA.
These will compete for the sequencing products and reduce the
effective signal. Use the volumes of ethanol recommended in the
protocol or calculate the volume that will yield a final
concentration of 70% ethanol.
5. An inappropriate salt was used to precipite the reaction products,
or the volume of salt used was incorrect. Use the ammonium
acetate included in the kit since the protocol has been optimized
with this salt.
6. The formamide loading solution was old. Aqueous solutions of
formamide ionize over time to produce ammonium formate
which increases the ionic strength of the buffer and reduces the
efficiency of injection. Using a 100% low conductivity formamide
stock, prepare a fresh solution of 70% formamide containing
0–1 mM EDTA and store at 4°C.
7. The DNA preparation was impure. Repeat the reaction using the
Control DNA supplied in the kit.
8. The primer or template contained excess EDTA. Resuspend
both primer and template in water or in dilute buffer containing
< 0.1 mM EDTA.
9. Either the quantity of template DNA or the number of cycles used
for amplification was insufficient. Increase either the amount of
DNA used in the reaction or the number of cycles.
10. The annealing temperature was too high for the primer being
used. Use a lower annealing temperature for cycling.
3211. Too little primer was used. The recommended amount of primer
is 5 pmol per reaction.
12. The sequence of the primer was inappropriate, forming dimers
or hairpins which can interfere with annealing. Change the
primer sequence.
13. The wrong volume of premix was used. The reagents are
carefully formulated to work optimally with 8 μl of premix in a
20 μl reaction volume or 4 μl of premix in a 10 μl reaction
volume. This ratio MUST be adhered to for optimal results. No
other configuration is recommended or supported.
14. Residual salt was present in the samples. This can affect the
ionic strength of the sample and interfere with electrokinetic
injection. If products of the sequencing reation were purified
using spin columns, confirm that they were eluted in water. Some
preparations of size exclusion chromatography media are pre-
swollen in a salt-containing buffer and must be washed several
times with water to remove the salt. In some cases, it might
be necessary to wash the dry media several times to remove
residual ions that can interfere with injection.
15. The template DNA was of poor quality. Contaminants (salt,
protein) can decrease the efficiency of electrokinetic injection.
High quality DNA prevents downstream sequencing problems.
Problem: Extensions appear short with read-length limited to
< 350 bases.
Possible causes/solutions
1. Too much template DNA was included in the sequencing reaction.
In some cases, the use of too much template, especially PCR
product DNA, can exhaust the supply of dye terminators in the
reaction. If this occurs, the sequence will suddenly fade before
reaching 350 bases in length. This problem is especially prevalent
33if excess primer is also present. Use < 1 pmol of template DNA
and 5 pmol of primer for each sequence. By using less template,
the concentration of any potential contaminant is also reduced.
2. The run voltage was too high. Limit the run voltage to ≤ 9 kV.
3. The extension step incubation period was too short. Increase the
duration of the extension step in the cycling program to
2–4 minutes.
Problem: Late signal-starts and broad, poorly resolved peaks are
prevalent in the sequences.
Capillary overloading that disrupts capillary current most often
causes late appearance of the primer peak and poorly resolved
sequencing fragment peaks. Overloaded samples frequently
produce low signals because the peaks are broad and diffuse, and it
is common to misdiagnose overloading as insufficient signal. Under
optimal conditions, detection of all samples should begin within a
few minutes of each other.
Possible causes/solutions
1. Excessive template DNA was used in the sequencing reaction and
carried over into the capillary upon electrokinetic sample injection.
Template DNA molecules compete with sequencing products for
injection, resulting in late starts and poorly resolved peaks. Use
less template in the sequencing reaction. To determine the
optimal amount of template, perform a titration of template
over a 50-fold range (0.2, 0.5, 1, 2, 5, and 11 μl, for example). This
titration can be accomplished easily in a single run with several
templates and control DNA.
2. The injection conditions were not optimal. Confirm that the
recommended injection conditions were used. Change the
injection conditions by reducing the duration of injection three-
fold.
343. The injection voltage was too high. Reduce the voltage to 2–3 kV.
4. Insufficient loading solution was used. Resuspend sequencing
reaction products in a larger volume of loading solution,
e.g. 20, 50 or 100 μl.
5. If the sequencing reaction products are in water, evaporate the
samples to dryness, resuspend in loading solution and then inject.
Problem: Localized broad peaks or very tall early peaks—
terminator blobs—are prevalent in the sequences.
Possible causes/solutions
1. Residual terminators were not eliminated from the samples.
Carefully follow the protocol (Step 2) for post-reaction cleanup.
Problem: Peak spacing changes during the run giving rise to the
“accordion effect”.
Possible causes/solutions
1. Samples were near the limits of overloading. Follow the
suggestions to avoid overloading described above within the
section “Late signal starts and broad, poorly resolved peaks are
prevalent in the sequences”.
Problem: Sequences are noisy or double sequences are present.
Possible causes/solutions
1. The annealing temperature of the sequencing reaction was too
low. Either increase the annealing temperature or eliminate it
completely for cycling between 95°C and 60°C. The effective
annealing temperature of primers is higher with DYEnamic ET Dye
Terminator Cycle Sequencing Kit for the MegaBACE DNA Analysis
Systems than with other terminator sequencing products.
If problems persist, please contact GE Healthcare’s Technical Service
for assistance.
3510. References
1. Sanger, F. et al., Proc. Nat. Acad. Sci. USA 74, 5463–5467 (1977).
2. Prober, J. M. et al., Science 238, 336–341 (1987).
3. Lee, L. G. et al., Nucleic Acids Research 20, 2471–2483 (1992).
4. Ju, J. et al., Proc. Nat. Acad. Sci. USA 92, 4347–4351 (1995).
5. Tabor, S. and Richardson, C. C., Proc. Nat. Acad. Sci. USA 84,
4767–4771 (1987).
6. Tabor, S. and Richardson, C. C., J. Biol. Chem. 264, 6447–6458
(1989).
7. Tabor, S. and Richardson, C. C., Proc. Nat. Acad. Sci. USA 92,
6339–6343 (1995).
8. Huibregtse, J. M. and Engelke, D. R., DNA and Protein Engineering
Techniques 1, 39–41 (1988).
9. McMahon, G. et al., Proc. Nat. Acad. Sci. USA 84, 4974–4978
(1987).
10. Carothers A. M. et al., Biotechniques 7, 494–496, 498–499 (1989).
11. Murray, V., Nucleic Acids Research 17, 8889 (1989).
12. Levedakou, E. N. et al., Biotechniques 7, 438–442 (1989).
13. Lee, J. S., DNA Cell Biol. 10, 67–73 (1991).
14. Messing, J., Methods in Enzymology 101, 20–78 (1983).
15. Mead, D. A. and Kemper, B. in Vectors: A Survey of Molecular
Cloning Vectors and Their Uses, Butterworth Publishers,
Massachusetts USA (1986).
16. Dente, L. et al., Nucleic Acids Research 11, 1645–1655 (1983).
17. Holmes, D. S. and Quigley, M., Anal. Biochem. 114, 193–197
(1981).
3618. Birnboim, H. C. and Doly, J., Nucleic Acids Research 24, 1513–1523
(1979).
19. Lizardi, P. et al., Nat. Genet. 19:225–32 (1998).
3738
39
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