GE 33, 79770, 188403, 79760, AH9539 User Manual
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Thermo Sequenase
Radiolabeled Terminator
Cycle Sequencing Kit
Product Number 79750, 50 reactions
79760, 100 reactions
79770, 500 reactions
Product Number 188403 includes:
79750, 50 reactions
AH9539,
terminators
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P-labeled
STORAGE
Store at -15°C to -30°C.
Warning: For research use only. Not
recommended or intended for diagnosis of
disease in humans or animals. Do not use
internally or externally in humans or animals.
CONTENTS
Components of the Kit .......................................................................................3
Quality Control....................................................................................................4
Safety Warnings and Precautions ..............................................................4 , 2 6
Introduction.........................................................................................................5
Materials Not Supplied....................................................................................... 8
Protocol ...............................................................................................................9
Supplementary Information.............................................................................11
General guidelines.........................................................................................11
Preparation of template DNA.........................................................................12
Cycle conditions and template quantity.........................................................12
Cycling temperatures.....................................................................................12
Number of cycles and quantity of template ...................................................1 3
Designing a new sequencing primer .............................................................13
Sequencing PCR products ............................................................................15
Elimination of compressions..........................................................................15
Reading farther from or closer to the primer .................................................17
Denaturing gel electrophoresis......................................................................18
Troubleshooting ...............................................................................................20
Control DNA Sequence ....................................................................................22
References ........................................................................................................23
Related Products ..............................................................................................24
Contact Information .........................................................................................25
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COMPONENTS OF THE KIT
The solutions included in the Thermo Sequenase™ Radiolabeled Terminator
Cycle Sequencing Kit have been carefully prepared to yield the best possible
sequencing results. Each reagent has been tested extensively and its
concentration adjusted to meet USB™ standards. It is strongly recommended
that the reagents supplied in the kit be used as directed.
The following solutions are included in the kit:
Thermo Sequenase DNA Polymerase: 4U/µl, 0.0006U/µl
acidophilum
inorganic pyrophosphatase**; 50mM Tris•HCl, pH 8.0, 1mM
dithiothreitol (DTT), 0.1mM ethylenediamine tetraacetic acid (EDTA), 0.5%
Tween™-20, 0.5% Nonidet™ P-40, 50% glycerol
Reaction Buffer (concentrate): 260mM Tris•HCl, pH 9.5, 65mM MgCl
dGTP Nucleotide Master Mix: 7.5µM dATP, dCTP, dGTP, dTTP
dITP Nucleotide Master Mix: 7.5µM dATP, dCTP, dTTP, 37.5µM dITP
Stop Solution: 95% formamide, 20mM EDTA, 0.05% bromophenol blue, 0.05%
xylene cyanol FF
Control DNA: double-stranded pUC18, 0.02µg/µl
Control Primer (-40 M13 forward; 23-mer): 2.0pmol/µl
5'-GTTTTCCCAGTCACGACGTTGTA-3'
This kit and all the enclosed reagents should be stored at -15°C to -30°C (NOT
in a frost-free freezer). Keep all reagents on ice when removed from storage for
use. The kit can conveniently be stored at 2°C to 4°C for periods of up to 3
months with no loss of performance, but this should be avoided if it is expected
that the reagents will not be completely consumed within 3 months.
Note: The formulation of Thermo Sequenase DNA polymerase in this kit
necessitates the use of a glycerol tolerant
8
DNA sequencing gel. See
‘Supplementary Information, denaturing gel electrophoresis’ section.
Thermoplasma
2
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P-labeled Terminators: A package of four 33P-labeled terminators must be
purchased for use with the kit. They may be ordered separately from GE Healthcare
using product number AH9539. In the US, the terminators
may be ordered together with the sequencing kit from USB using product
number 188403.
ddGTP, 0.3µM [α-33P]ddGTP (1500Ci/mmol, 450µCi/ml), Redivue™
ddATP, 0.3µM [α-33P]ddATP (1500Ci/mmol, 450µCi/ml), Redivue
33
ddTTP, 0.3µM [α-
P]ddTTP (1500Ci/mmol, 450µCi/ml), Redivue
ddCTP, 0.3µM [α-33P]ddCTP (1500Ci/mmol, 450µCi/ml), Redivue
3
Redivue nucleotides can be stored at 4
at a constant -20
prevent evaporation of these small volumes of material. Tightly cap the
vials after use. Store at -20°C between uses if frequency of use is less
than every 1-3 days. If condensation is observed on the walls of the vial or
in the cap, return the liquid to the bottom of the vial and mix well before
use.
°C if longer storage is desired. Care must be taken to
°C for up to 1 week after receipt, or
QUALITY CONTROL
All kit batches are functionally tested using 33P labeled terminators and pUC18
double-stranded DNA template as described in this protocol. Release
specifications are based on sequence length, band intensity and sequence
quality. The sequence must be visible up to 300 base pairs on a standardized
gel with less than 24 hours exposure. The sequence must also be free of
background bands strong enough to interfere with sequence interpretation.
SAFETY WARNINGS AND PRECAUTIONS
Warning: For research use only. Not recommended or intended for
diagnosis of disease in humans or animals. Do not use internally or
externally in humans or animals.
Caution: This product is to be used with radioactive material. Please follow the
manufacturer’s instructions relating to the handling, use, storage, and disposal
of such materials.
Warning: Contains formamide. See ‘Material Safety Data Sheet’ on page 26.
All chemicals should be considered as potentially hazardous. We therefore
recommend that this product is handled only by those persons who have been
trained in laboratory techniques and that it is used in accordance with the
principles of good laboratory practice. Wear suitable protective clothing such as
a lab coat, safety glasses, and gloves. Care should be taken to avoid contact
with skin or eyes. In the case of contact with skin or eyes, wash immediately
with water (see ‘Material Safety Data Sheet’ for specific advice).
4
INTRODUCTION
This sequencing kit combines two revolutionary innovations for sequencing
DNA using radioactive labels. First, the label is incorporated into the DNA
sequencing reaction products by the use of four [α-
(ddNTP) terminators (G,A,T,C). The labeled ddNTPs are more efficient for
labeling sequencing experiments than other labeled nucleotides because they
specifically label only the properly terminated DNA chains. Also, since
prematurely terminated chains are not labeled, ‘stop’ artifacts and most
background bands are eliminated. As an additional benefit, the absence of
artifact bands allows the routine use of dITP, which can eliminate even very
strong compression artifacts.
The second innovation is the use of Thermo Sequenase DNA polymerase
This enzyme has been engineered to efficiently incorporate dideoxynucleotides,
allowing the use of very low amounts of isotope ([α-33P]ddNTP) for the
termination reactions. Thermo Sequenase DNA polymerase is also
thermostable and performs very well in convenient and sensitive cycle or noncycle sequencing methods. This polymerase produces very uniform band
intensities (with dGTP), so mixed sequences (such as those of heterozygotes)
can be easily identified.
Thus, the kit offers:
• Clean, background-free sequences
• Complete elimination of compressions
• Efficient use of labeled nucleotides, less than 1µCi per sequence
• Convenient single-step protocol
• Uniform band intensities for identification of mixed sequences (
heterozygotes)
• Sensitive cycle-sequencing protocols for sequencing 20fmol or less of
template
• Overnight exposures with ordinary autoradiography film—same day results
possible with fast films
• Exceptionally easy-to-read sequences
33
P for sharp autoradiogram resolution
•
• Sample storage for 1-2 days prior to running on gel
33
P]dideoxynucleotide
e.g.
‡
.
5
Chain termination sequencing
This kit is designed to eliminate sequencing artifacts such as stops (or BAFLs—
bands across four lanes) and background bands. BAFLs can result from the
enzyme pausing at regions of secondary structures in GC-rich templates,
producing prematurely aborted primer extension products of the same length in
all four termination reactions. Background bands can be caused by primer
extensions aborting prematurely at random positions, such as when a template
is rich in a certain base and the complementary nucleotide in the reaction
becomes depleted.
Traditional chain termination sequencing methods (1) involve the synthesis of a
in vitro
DNA strand by a DNA polymerase
template. Synthesis is initiated at the site where a primer anneals to the
template. Elongation of the 3' end of the annealed primer is catalyzed by a DNA
polymerase in the presence of 2'-deoxynucleoside-5'-triphosphates (dNTPs),
and is terminated by the incorporation of a 2',3'-dideoxynucleoside-5'triphosphate nucleotide analog (ddNTP) that will not support continued DNA
elongation (hence the name ‘chain termination’). Four separate reactions, each
with a different ddNTP, (ddG, ddA, ddT, or ddC), give complete sequence
information. A radiolabeled dNTP (2,3) or primer is normally included in the
synthesis, so the labeled chains of various lengths can be visualized after
separation by high-resolution gel electrophoresis (4,5). In this kit, a radioactive
label is incorporated into the sequencing reaction products at the 3' end by the
use of an [α-
33
P]ddNTP, thus ensuring that only properly terminated DNA
strands are labeled and are visible in the sequence. This results in a cleaner,
more reliable and easier to read sequence with fewer background bands and
virtually no BAFLs.
The accuracy and readability of the sequence obtained depends strongly on the
properties of the polymerase used for chain termination. Some polymerases,
such as Sequenase™ Version 2.0 DNA polymerase, generate much more
uniform, readable bands than others like Klenow and
(6,7,8). Thermostable polymerases, such as
multiple rounds (cycles) of DNA synthesis, generating stronger signals. Tabor
and Richardson (9) have discovered that DNA polymerases can be modified to
accept dideoxynucleotides as readily as the normal deoxynucleotide substrates.
Using this technology, a new DNA polymerase for DNA sequencing was
developed. This enzyme, called Thermo Sequenase DNA polymerase, is
thermostable and possesses many of the excellent DNA sequencing qualities of
Sequenase DNA polymerase. The properties of this DNA polymerase include
activity at high temperature and absence of associated exonuclease activity.
Like Sequenase DNA polymerase, derived from T7 bacteriophage, it readily
using a single-stranded DNA
Taq
DNA polymerase
Taq
polymerase, can be used for
6
uses dideoxynucleoside triphosphates, generating uniform band intensities in
sequencing experiments (with dGTP). These properties make the enzyme ideal
for generating high-quality DNA sequences using cycle-sequencing methods. It
is stable at 90°C for at least 1 hour and retains 50% of its activity when
incubated at 95°C for 60 minutes. The Thermo Sequenase polymerase in this
kit combines the advantages of both Sequenase DNA polymerase and
Taq
DNA polymerase. It produces bands (with Mg2+) that are nearly as uniform as
2+
those produced with Sequenase DNA polymerase with Mn
Taq
thermostable like
DNA polymerase.
(10), yet is
Cycle sequencing is the name given to the process of using repeated cycles of
thermal denaturation, primer annealing, and polymerization to produce greater
amounts of product in a DNA sequencing reaction. This amplification process
employs a single primer so the amount of product DNA increases linearly with
the number of cycles. (This distinguishes it from PCR* which uses 2 primers so
that the amount of product can increase exponentially with the number of
cycles.)
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The earliest examples of cycle sequencing used
P-labeled primers and a nonthermostable polymerase which was added after each denaturation cycle
(11,12). Later improvements included the use of thermostable
Taq
polymerase
(13,14) and the use of alpha-labeled dNTPs in place of the labeled primer
using mixtures of nucleotides similar to those used originally by Sanger (15,16).
The labeled-primer methods make efficient use of
32
as little as 4µCi of [γ-
products were less efficient, requiring either 10µCi of [α-
35
[α-
S]dATP for a sequence. This is a consequence of the relatively low specific
P]ATP (14). The methods using internally-labeled
32
P giving a sequence with
33
P]dATP or 20µCi of
radioactivity and the small number of labeled bases in short product molecules.
33
This kit makes very efficient use of [α-
33
P per sequence. Cycle sequencing is necessary with this kit when using less
P]ddNTP, requiring less than 1µCi of
than 0.2-0.5pmol of template DNA. Non-cycle (or very few cycle) protocols may
be used with more than ~0.5pmol of template.
*See license information on back cover.
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MATERIALS NOT SUPPLIED
Necessary reagents:
Water—Only deionized, distilled water should be used for the sequencing
reactions.
Specialized sequencing primers—Some sequencing projects will require the
use of primers which are specific to the project. For most sequencing
applications, 0.5-2.5pmol of primer should be used for each set of sequencing
reactions. Always determine the concentration of the primer by reading the
optical density at 260nm (OD
concentration (pmol/µl) is given by the following formula:
Concentration (pmol/µl)=OD
Gel reagents—Sequencing gels should be made from fresh solutions of
acrylamide and bis-acrylamide. Other reagents should be electrophoresis grade
materials. For convenience, RapidGel™ gel mixes are strongly recommended.
RapidGel-XL formulations yield up to 40% more readable sequence per gel.
See ‘Related Products’ section for range of USB Ultrapure gel products.
Necessary equipment:
Liquid handling supplies such as vials, pipettes and a microcentrifuge—All
sequencing reactions are run in plastic microcentrifuge tubes (typically 0.5ml)
suitable for thermal cycling.
Electrophoresis equipment—While standard, non-gradient sequencing gel
apparatus is sufficient for much sequencing work, the use of field-gradient
(‘wedge’) or salt-gradient gels will allow much greater reading capacity on the
gel (4,5,17). A power supply offering constant voltage operation at 2000V or
greater is essential.
Gel handling—For 33P sequencing, a large tray for washing the gel (to remove
urea) and a gel drying apparatus are highly recommended. For best results,
gels containing 33P must be exposed dry in direct contract with the film at room
temperature.
Autoradiography—Any large format autoradiography film such as the
BioMax™ MR, and a large film cassette.
Thermal cycler—Sequencing will require thermally cycled incubations between
50°C and 95°C (1-100 cycles).
). If the primer has N bases, the approximate
260
/(0.01 x N) where N is the number of bases.
260
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PROTOCOL
1. Termination mixes—Prepare the termination mixes on ice. Mix 2µl of
Nucleotide Master Mix (either dGTP or dITP—see note below) and 0.5µl of
33
P]ddNTP (G, A, T, or C—one of each per sequence) to produce a
[αtermination mix for each ddNTP. Label, fill and cap four tubes (‘G’, ‘A’, ‘T’,
‘C’) with 2.5µl of each termination mix. It is more accurate and convenient
to prepare batches of termination mixes sufficient for all sequences to
be performed, then dispense 2.5µl from this batch to each vial for the
termination reactions. It is recommended that these batches of termination
mixes be made up routinely.
To prepare termination mixes for (n) reactions, mix:
GATC
Nucleotide Master Mix (2 x n)µl (2 x n)µl (2 x n)µl (2 x n)µl
33
P]ddNTP (0.5 x n)µl (0.5 x n)µl (0.5 x n)µl (0.5 x n)µl
[α-
Total (2.5 x n)µl (2.5 x n)µl (2.5 x n)µl (2.5 x n)µl
Note: The termination tubes can be left uncapped until all reagents have
been added if the tubes are kept on ice and the reaction mixture is added
within a few minutes. For determination of new sequences, or of sequences
with high G-C content, the dITP Nucleotide Master Mix is recommended. This
will eliminate all compression artifacts but will result in somewhat uneven
band intensities, especially in the ‘G’ lane. When perfectly uniform band
intensities are desired, such as when examining sequences from potentially
heterozygous individuals, the dGTP Nucleotide Master Mix should be used.
2. Reaction mixture:
For multiple (n) reactions with different primers and/or templates, prepare a
n+1 batch of reaction buffer, water, polymerase and aliquot; then add the
unique primer and/or template in the appropriate concentration and volume to
the aliquots.
Reaction Buffer 2µl
DNA _µl*(50-500ng or 25-250fmol)
Primer _µl*(0.5-2.5pmol)
O_µl (To adjust total volume to 20µl)
H
2
Thermo Sequenase polymerase (4U/µl) 2µl (8 units polymerase—add LAST)
Total 20µl
*For the control reaction, use 10µl of control DNA and 1µl of control primer.
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