GE Recording Equipment 100 reactions 79770 User Manual

Thermo Sequenase

RadiolabeledTerminator

Cycle Sequencing Kit

Product Number 79750, 50 reactions

79760, 100 reactions

79770, 500 reactions

Product Number 188403 includes:

79750, 50 reactions

AH9539, ³³P-labeled

terminators

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.

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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 Thermoplasma

•

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 MgCl₂

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 tolerant⁸DNA sequencing gel. See

‘Supplementary Information, denaturing gel electrophoresis’ section.

³³P-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

ddTTP, 0.3µM [α-³³P]ddTTP (1500Ci/mmol, 450µCi/ml), Redivue

ddCTP, 0.3µM [α-³³P]ddCTP (1500Ci/mmol, 450µCi/ml), Redivue

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Redivue nucleotides can be stored at 4°C for up to 1 week after receipt, or

at a constant -20°C if longer storage is desired. Care must be taken to

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.

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).

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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-³³P]dideoxynucleotide

(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 non-

cycle 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 (e.g.

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

•

³³P for sharp autoradiogram resolution

• Sample storage for 1-2 days prior to running on gel

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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

DNA strand by a DNA polymerase in vitro using a single-stranded DNA

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-³³P]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 Taq DNA polymerase

(6,7,8). Thermostable polymerases, such as Taq polymerase, can be used for

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

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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

those produced with Sequenase DNA polymerase with Mn²⁺(10), yet is

thermostable like Taq DNA polymerase.

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.)

The earliest examples of cycle sequencing used ³²P-labeled primers and a non-

thermostable 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 ³²P giving a sequence with

as little as 4µCi of [γ-³²P]ATP (14). The methods using internally-labeled

products were less efficient, requiring either 10µCi of [α-³³P]dATP or 20µCi of

[α-³⁵S]dATP for a sequence. This is a consequence of the relatively low specific

radioactivity and the small number of labeled bases in short product molecules.

This kit makes very efficient use of [α-³³P]ddNTP, requiring less than 1µCi of

³³P per sequence. Cycle sequencing is necessary with this kit when using less

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 (OD₂₆₀). If the primer has N bases, the approximate

concentration (pmol/µl) is given by the following formula:

Concentration (pmol/µl)=OD₂₆₀/(0.01 x N) where N is the number of bases.

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).

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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

[α-³³P]ddNTP (G, A, T, or C—one of each per sequence) to produce a

termination 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:

Nucleotide Master Mix

(2 x n)µl

[α-³³P]ddNTP

(0.5 x n)µl (0.5 x n)µl (0.5 x n)µl (0.5 x n)µl

––––––––– ––––––––– ––––––––– –––––––––

(2.5 x n)µl (2.5 x n)µl (2.5 x n)µl (2.5 x n)µl

Total

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

DNA

Primer

2µl

_µl* (50-500ng or 25-250fmol)

_µl* (0.5-2.5pmol)

H₂O

_µl (To adjust total volume to 20µl)

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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3. Cycling termination reactions

Transfer 4.5µl of reaction mixture (prepared in step 2) to each termination

tube (‘G’, ‘A’, ‘T’ and ‘C’) from step 1. Mix well and overlay with 10-20µl of

mineral oil (if needed). Cap and place the tube in the thermal cycling

instrument.

Note: When sequencing single-stranded DNA, the primer may anneal to the

template with reduced specificity while the tubes are on ice, and extension of

these primers can occur as the thermal cycler heats up during the first cycle.

To minimize nonspecific extension products, the cycler can be pre-heated to

85-95°C or pre-cooled to 4°C.

4. Start the cycling program. Note: The specific cycling parameters used will

depend on the primer sequence and the amount and purity of the template

DNA. For the primers included in the kit and the suggested amount of purified

DNA (25-250fmol), cycle 30-60 times as follows:

dGTP

dITP

95°C, 30s

55°C, 30s

50°C, 30s

72°C, 60-120s

60°C, 5-10min

(typically 30 cycles taking 2-3hr) (typically 30 cycles taking 3-5hr)

Fewer (1-10) cycles may produce better results when using 250-500fmol DNA.

5. Add 4µl of Stop Solution to each of the termination reactions, mix thoroughly

and centrifuge briefly to separate the oil from the aqueous phase.

Alternatively, remove 6µl from each termination reaction and transfer to a

fresh tube containing 3-4µl of Stop Solution. Samples should be kept on ice

for same day loading or may be stored frozen up to 3 days before loading onto

gel.

6. When the gel is ready for loading, heat the samples to 70°C for 2-10 minutes

and load immediately on the gel—3-5µl in each gel lane. Note: Heating in

open vials will promote evaporation of water from the formamide-reaction

mixture. This is not normally necessary, but will increase the signal by

concentrating the isotope and will promote more complete denaturation of the

DNA. This may improve results when using older ³³P-ddNTPs. Avoid complete

evaporation to dryness by prolonged heating.

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SUPPLEMENTARY INFORMATION

General guidelines

• Since the popular multiple cloning sites all derive from similar sequences,

one primer can serve for the sequencing of insert DNA in most of the

common vectors. Among the vectors compatible with the primer supplied in

the Thermo Sequenase radiolabeled terminator cycle sequencing kit are

M13mp8, M13mp9, M13mp12, M13mp13, M13mp18, M13mp19, mWB2348,

mWB3295, mWB3225, pUC18, pUC19, and virtually any vector featuring

blue/white screening with β-galactosidase activity.

• Good sequences can be obtained using as little as 0.05µg of M13 DNA,

0.1µg of plasmid DNA, or 50fmol of PCR product. Mix reagents by gently

‘pumping’ the pipettor. The total volume of the reaction mix should be 20µl—

the volumes of DNA and primer added will depend on their concentration.

Adjust the amount of distilled water so that the total volume of DNA, primer

and water is 16µl.

• The specific cycling parameters used will depend on the primer sequence

and the amount and purity of the template DNA. See ‘Supplementary

Information, cycle conditions and template quantity’.

• The dGTP Nucleotide Master Mix should be used if the sequence is already

known to be free of compression artifacts and the benefits of uniform band

intensities are desired. The uniform band intensities can aid in finding

heterozygotes or in other cases where mixed sequence may be present. If

compressions are a problem when using dGTP, gels containing formamide

can be used as described in the ‘Supplementary Information, denaturing gel

electrophoresis’ section of this booklet.

• For running sequences where compressions are a problem, the dITP

Nucleotide Master Mix included in this kit can be substituted for the dGTP

Nucleotide Master Mix. See ‘Supplementary Information, elimination of

compressions’ section for details. Note: When using dITP, use an ‘extension’

temperature of 60°C with a duration of at least 4 minutes.

• Whenever possible, tubes should be kept capped and on ice to minimize

evaporation of the small volumes employed. Additions should be made with

disposable-tip micropipettes and care should be taken not to contaminate

stock solutions. The solutions must be thoroughly mixed after each addition,

typically by ‘pumping’ the solution two or three times with a micropipette,

avoiding the creation of air bubbles. At any stage where the possibility exists

for some solution to cling to the walls of the tubes, the tubes should be

centrifuged. With care and experience these reactions can be set-up in 15-20

minutes.

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Preparation of template DNA

Since cycle sequencing can be performed using very little template DNA, only

very small amounts of detrimental impurities are likely to be carried along with

the DNA. Therefore, though still important, template purity may not be as crucial

for cycle sequencing as it is for non-cycle sequencing.

Preparation of single-stranded template DNA

Single-stranded template DNA of good purity is essential for excellent

sequencing results. Several popular plasmid cloning vectors contain the same

lac-derived cloning region as the M13mp vectors and a single-stranded phage

replication origin. Production of single-stranded DNA from these vectors is

similar to that of the M13 phage and the single-stranded DNA produced can

also be used as template for sequencing.

Preparation of double-stranded plasmid DNA

Sequencing double-stranded templates with the Thermo Sequenase

Radiolabeled Terminator Cycle Sequencing Kit works effectively with no

changes in the reaction protocol. Alkaline denaturation is not required for

plasmid DNA templates. For best results, purified plasmid DNA should be

used—CsCl gradients, PEG precipitation, adsorption to glass, columns, and

other common DNA purification methods all produce suitable DNA. (However,

since such small quantities of DNA are added to the reactions, even impure

DNA samples can sometimes yield acceptable sequence data.) There are many

popular protocols for purifying plasmid DNA from 2-10ml cultures. We have had

consistent success with ‘boiling’ (21) and ‘alkaline’ (22) mini-prep methods.

Cycle conditions and template quantity

The temperatures used for cycling the termination reactions should be

determined from the characteristics of the sequencing primer, the template, and

the length of the termination product desired. The number of cycles required will

depend on the quantity and quality of the template DNA used. The following

guidelines should assist in choosing cycling parameters.

Cycling temperatures

The melting temperature of the primer should be kept in mind when choosing

cycle temperatures. The control primer included in the kit is moderately long (23

bases) with 50% G/C content. The melting temperature of this primer is ~73°C

under sequencing reaction conditions, and excellent results are achieved by

cycling between 60°C and 95°C. The duration of the steps does not seem to be

critical, and even brief pauses (1-10 seconds) at these temperatures seem to

be effective (except with dITP as described above).

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As another example, when using the universal -40 17-mer, which has a melting

temperature of about 50°C, cycling between 45°C and 95°C is effective. If in

doubt, choose a wide temperature range with pauses (15-30 seconds) at the

extremes of temperature.

The termination reaction cycles should always have a denaturation temperature

of 95-98°C (however, avoid extended steps at 98°C since at this temperature

the enzyme has a half-life of less than one hour). Since the optimum

temperature for polymerization is about 70-75°C, 72°C is a good choice for the

termination step (except when using dITP, which requires a maximum

temperature of 55-60°C). An annealing step (e.g. <60°C) is required only with

primers less than ~24 bases.

Number of cycles and quantity of template

The number of cycles required will primarily depend on the amount of template

DNA (in fmols) used for sequencing. It will also depend on the purity of the

DNA, and the sensitivity of autoradiographic detection. The minimum quantities

of highly-purified DNA which we have been able to sequence using these

methods are about 5fmol of M13mp18 DNA and about 15fmol of pUC18 DNA.

(For routine sequencing, we recommend 25fmol of M13 and 75fmol of plasmid

DNA). When sequencing very small amounts of template, it has been observed

that the number of cycles has a strong influence on sequence intensity.

Increasing the number of cycles from 30 to 60 will increase the signal

significantly when using less than ~50fmol of template DNA, whereas

increasing the number of cycles with more than ~100fmol is of little benefit, and

may even produce background sequence. So in general, use more cycles when

template amounts are limited. Also, a modest improvement can sometimes be

achieved by increasing the amount of primer 2-5 fold. It is undesirable to use

too much template as the result will be a shortened sequence extension. Figure

1 shows the result of increasing template quantities to an excess.

Designing a new sequencing primer

The length of the primer (and its sequence) will determine the melting

temperature and specificity. For the cycling temperatures normally used, the

primer should be about 18-25 nucleotides long. It is also a good idea to check

the sequence of the primer for possible self-annealing (dimer formation could

result) and for potential ‘hairpin’ formation, especially those involving the 3' end

of the primer. Finally, check for possible sites of false priming in the vector or

other known sequence if possible, again stressing matches which include the 3'

end of the primer.

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0.5pmol

1pmol

2pmol

8pmol

300 bases

150 bases

Figure 1. Excess template DNA can reduce sequence extension lengths. In cases where

2pmol or more template DNA are sequenced, the supply of nucleotides can be exhausted

before extensions reach suitable length for optimal sequencing. These sequences were

run using up to 16µg (8pmol) of M13mp18 DNA template.

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Sequencing PCR Products

The products of Polymerase Chain Reaction (PCR) can have structures which

make them difficult to sequence. One of the most common problems associated

with sequencing of PCR products is the presence of stops or BAFLs, where the

sequence pauses or stops at artifactual ends in the template (actually the ends

of truncated PCR product). This kit incorporates label by way of a radiolabeled

dideoxy terminator so that only the fragments which were properly terminated

are visible in the sequence. No labeled bands are formed at ‘ends’ in the

template, eliminating many of these artifacts and enabling sequences to extend

to essentially the last base of a PCR product. Artifacts caused by appearance of

double-stranded PCR product on denaturing gels are similarly eliminated since

they are not labeled. Following is information which should assist in producing

high quality, reliable sequence information even with PCR product templates

which have been very difficult to sequence with standard methods.

It is essential that PCR products are of high quality and quantity in order to

obtain high quality sequence information. Problems with high background, low

signal intensity and ambiguities can often be traced to the PCR step. Not every

PCR will yield a product which can be sequenced. Analysis of the PCR product

on agarose gels and optimization of the PCR may be necessary to obtain

quality sequences.

Enzymatic pre-treatment of PCR products

The key step in this method for sequencing PCR products consists of treating

the PCR product with a combination of Exonuclease I and Shrimp Alkaline

∞

Phosphatase to eliminate any primer or dNTPs which were not incorporated

into the PCR product. These enzymes are available from USB in a reagent pack

(70995) or pre-mixed (ExoSAP-IT™, 78200) with detailed protocols for their

usage. It is recommended that this enzymatic clean-up of the PCR product be

used with this sequencing method.

Elimination of compressions

Some DNA sequences, especially those with dyad symmetries containing dG

and dC residues, are not fully denatured during electrophoresis. When this

occurs, the regular pattern of migration of DNA fragments is interrupted; bands

are spaced closer than normal (compressed together) or sometimes farther

apart than normal and sequence information is lost. The substitution of a

nucleotide analog (dITP) for dGTP which forms weaker secondary structure has

been successful in eliminating most of these gel artifacts (18, 19). Two

examples are shown in figure 2 in the sequences run with dGTP.

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dGTP

dITP

dGTP

dITP

→

Figure 2. Compression artifacts can be eliminated using dITP in place of dGTP without

interference by stops or other artifact bands. Shown are two severely ‘compressed’

regions of secondary structure (see arrows). The sequences run using dITP in place of

dGTP are accurate and unambiguous.

A suitable nucleotide mixture containing dITP is included in the kit for use with

templates prone to gel compression artifacts. To use dITP simply substitute the

dITP Nucleotide Mix for the dGTP Nucleotide Mix. All other aspects of the

sequencing protocol remain unchanged except that when using dITP, reduce

the termination temperature from 72°C to 60°C and increase the time to

approximately 5 minutes or longer (see figure 3). The use of dITP will result in

less uniform band intensities, but will completely resolve even the strongest

compressions. A 40% formamide gel will also eliminate almost all compressions

(see ‘Denaturing gel electrophoresis’ section).

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1min

4min

10min

20min

300 bases

200 bases

150 bases

Figure 3. Use of dITP requires longer extension times at 60°C. Shown are four

sequences of plasmid pUC18 obtained using cycles with 1, 4, 10 and 20 minute

extension steps in the cycles. Extension steps of 4-5 minutes or longer are necessary for

reading beyond 200 bases.

Reading farther from or closer to the primer

The termination mixes described in the protocol will typically yield sequencing

data from the first base to over 500 bases from the primer. This is as much

sequence as most users will be able to read using current standard

electrophoresis technology. If it is desired to obtain sequence >500 bases from

the primer, the dNTP:ddNTP ratios can be easily altered to shift the distribution

of sequencing reaction products by adding more dNTPs to the termination

reaction. Adding 3µl instead of 2µl dNTPs will increase the dNTP:ddNTP ratio

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by 50%, thus increasing the average extension length of each primer before a

ddNTP is incorporated. Conversely, adding 1µl of [α-³³P]ddNTP will decrease

the ratio by 50%, thus decreasing the average extension length of each primer.

Running sequencing gels which resolve more than 600 nucleotides requires

high quality apparatus, chemicals and attention to many details. While specific

instructions are beyond the scope of this manual, following are some general

guidelines: The gel should be loaded with 8 adjacent lanes (GATCGTAC or see

‘Supplementary Information, denaturing gel electrophoresis’ section) with a

sharkstooth comb and be run 4 to 10 times longer than usual. For this kind of

experiment, gradient (or ‘wedge’) gels or very long gels (80-100cm) are almost

a necessity. The highest resolution gels appear to be approximately 6-8%

acrylamide and are run relatively cool (40°C).

Denaturing gel electrophoresis

Under optimal gel electrophoresis conditions, 250-300 bases can be read from

the bottom of a standard size sequencing gel. The length of time the gel is run

will determine the region of sequence that is readable. Many factors can limit

the sequence information which can be determined in a single experiment.

Among these are the quality of reagents used, the polymerization, the

temperature of the gel during electrophoresis, and proper drying of the gel after

running. The greatest care should be given to the pouring and running of

sequencing gels. The specifics of running the electrophoresis will depend on

the apparatus used. The following suggestions for reagent compositions and

procedures are intended as guidelines. For specific instructions contact the

manufacturer of the gel apparatus used.

Gel electrophoresis reagents

This kit contains a prediluted enzyme mixture which contains a high glycerol

concentration, requiring the use of a glycerol tolerant gel buffer. The use of

other buffers such as TBE can result in severe distortion of sequencing bands

in the upper third of the gel. The following recipe is for typical sequencing gel

reagents.

Buffers

20X Glycerol Tolerant Gel Buffer (71949 or 75827)

Tris base

Taurine

Na₂EDTA 2H₂O

216g

72g

H₂O to 1000ml, filter (may be autoclaved)

This buffer can be used with samples containing glycerol at any concentration

(20). If gels seem to run a bit slower with this buffer at 1X strength, use it more

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dilute—approximately 0.8X strength. Be certain to run glycerol tolerant gels at

the same power (wattage) as TBE-buffered gels so the gel temperature is

normal.

10X TBE Buffer (70454)

Tris base

Boric acid

Na₂EDTA 2H₂O

108g

55g

9.3g

H₂O to 1000ml, filter (may be autoclaved)

This is the traditional sequencing gel buffer. It should NOT be used with the

polymerase supplied in this kit (Glycerol Tolerant Gel Buffer should be used).

Gel recipes (for 100ml of gel solution)

Standard gel

Gel conc. Acrylamide/

Urea 20X Gly. Tol. OR 10X TBE

Buffer

(%)

bis-acrylamide (7-8.3M) Gel Buffer

H₂O

5.7g/0.3g

7.6g/0.4g

5.7g/0.3g

7.6g/0.4g

42-50g

5ml*

~45ml

~40ml

10ml

Dissolve, adjust volume to 100ml with H₂O, filter and de-gas. When ready to

pour, add 1ml of 10% ammonium persulfate and 25µl TEMED (N, N, N', N'-

tetramethylethylenediamine).

*Use 4ml for faster gel migration.

Formamide gel (for resolution of compressions)

Gel conc. Acrylamide/ Urea* 20X Gly. Tol. OR 10X TBE

(%) bis-acrylamide (7M)

Gel Buffer

Buffer Formamide H₂O

5.7g/0.3g

7.6g/0.4g

5.7g/0.3g

7.6g/0.4g

42g

5ml

40ml

~10ml

~5ml

10ml

~5ml

*Warming to 35-45°C may be required to dissolve urea completely.

Adjust volume to 100ml with H₂O, filter and de-gas. When ready to pour add

1ml of 10% ammonium persulfate and 100-150µl TEMED. The temperature of

the mixture should be 25-35°C—warmer mixtures will polymerize too fast while

mixtures below 20°C may precipitate urea. They will require higher running

voltage and run slower than urea-only gels. Prior to drying, these gels should be

soaked in 5% acetic acid, 20% methanol to prevent swelling. For more detailed

information, refer to TechTip #200 available from USB Technical Support or the

Technical Library at usbweb.com.

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General guidelines for electrophoresis

1. Ultrapure or electrophoresis grade reagents should be used.

2. Sequencing gels should be made fresh. Store solutions no longer than one

week in the dark at 4°C. Commercial preparations of acrylamide gel mixes in

liquid or powder form (RapidGel gel mixes—see ‘Related Products’) should

be used according to manufacturers recommendations.

3. Gels should be prepared 2-20 hours prior to use, and pre-run for ~15

minutes.

4. When reading longer sequences, it is usually convenient to run gels

overnight with a timer-controlled power supply. Gel runs of 18-24 hours at

40-50 watts are often necessary for reading in the 400-600bp range.

5. Loading 8 adjacent lanes in a pattern that abuts all pairs of lanes (e.g.

GATCGTAC) aids reading closely spaced bands.

6. Gels should be soaked in 5% acetic acid, 15% methanol to remove the urea.

Soaking time depends on gel thickness. Approximate minimum times are 5

minutes for 0.2mm gels, 15 minutes for 0.4mm gels and 60 minutes for field

gradient (0.4-1.2mm wedge) or formamide gels. Drying should be done at

moderate temperature (80°C) to preserve resolution.

7. If RapidGel-XL is used, the gel does not need to be soaked. In fact, soaking

RapidGel-XL gels will cause swelling thereby affecting band resolution in the

final result.

8. For ³³P gels, autoradiography must be done with direct contact between the

dried gel and the emulsion side of the film. Gels dried without prior soaking

(leaving plastic-wrap on helps to prevent the film from sticking to the

incompletely-dried gels) will require longer drying and exposure times but

give sufficient resolution for most purposes.

9. Good autoradiography film can improve image contrast and resolution. We

recommend Kodak Biomax™ MR or Hyperfilm™-bmax autoradiography film.

10.In general, overnight to 36 hour exposures are sufficient when using fast film

such as Hyperfilm™-MP.

11.The use of tapered spacers (‘wedge' gels) improves overall resolution and

allows more nucleotides to be read from a single loading (4).

TROUBLESHOOTING

Problem

Possible causes and solutions.

Extensions appear short (read length limited to less than 200 bases)

1. If using dITP, increase time of extension step in cycles to 5-10 minutes and

decrease temperature to 60°C. See figure 2.

2. Too much template DNA. In some cases, the use of too much DNA,

especially PCR product DNA, can exhaust the supply of ddNTPs. Use less

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than 1pmol of template DNA for each sequence (0.25pmol per reaction).

See figure 1.

3. G-C rich template producing strong secondary structure. Try less DNA,

longer extension times, more cycles, more enzyme, 5% DMSO, or a 96°C

denaturation temperature.

Film blank or very faint

1. If using single-sided film, the emulsion side must be placed facing the dried

gel.

2. DNA preparation may be bad. Try the control DNA supplied in the kit.

3. Labeled dideoxynucleotide too old. Try longer exposure.

4. Some component missing.

5. Enzyme lost activity.

6. Insufficient template DNA or insufficient number of cycles. Try more DNA,

more cycles or longer film exposure.

7. Incorrect temperatures for primers used. Try a lower temperature for cycling

(e.g. 50°-95°C), especially when using dITP.

8. Incorrect termination time or temperature for dITP. Termination should be 5-

10 minutes at 55-60°C.

8. Too little primer used. The recommended amount of primer is 0.5-2.5pmol.

9. Primer bad. Some primers form dimers, hairpins etc., interfering with

annealing with the template. Try a different primer.

10.Wrong amounts of dNTP or [a-³³P]ddNTP used. Check volumes added.

11.Large excess of primer and DNA used. Check quantities added to reaction.

Bands faint near the primer

1. Too much dNTP or too little [α-³³P]ddNTP used. Check volumes added.

Bands smeared

1. Contaminated DNA preparation. Try control DNA. Thermo Sequenase DNA

polymerase is sensitive to salt concentration, especially above 75mM.

2. Gel may be bad. Gels should be cast with fresh acrylamide solutions and

should polymerize rapidly, within 15 minutes of pouring. Try running a

second gel with the same samples.

3. Gel run too cold. Sequencing gels should be run at 40-55°C.

4. Gel dried too hot or not flat enough to be evenly exposed to film.

5. Samples not denatured. Make sure samples are always heated to 70°C for

at least 2 minutes (longer in an air-filled heat block) immediately prior to

loading on gel. When re-loading a sample (e.g. for a second gel or a double-

loaded gel) the heating step should be repeated.

Bands appear across all 4 lanes

1. Gel compression artifacts. Sometimes a band in all 4 lanes indicates a

severe gel compression caused by secondary structures not completely

denatured during electrophoresis. If the gel has a region where the bands

are very closely spaced, followed by a region where the bands are widely

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spaced, a compression artifact is indicated. Try using the dITP reaction

mixture or a formamide gel.

Bands in 2 or 3 lanes

1. Heterogeneous template DNA (2 bands) caused by spontaneous deletions

arising during M13 phage growth. Try control DNA and limit phage growth to

less than 6-8 hours.

2. Insufficient mixing of reaction mixtures.

3. The sequence may be prone to compression artifacts in the gel.

Compressions occur when the DNA (usually G-C rich) synthesized by the

DNA polymerase does not remain fully denatured during electrophoresis. Try

using the dITP reaction mixture, or a 30-40% formamide gel.

If problems persist please contact USB Technical Support for assistance at

(800) 321-9322 or techsupport@usbweb.com in the United States. For your

authorized distributor and support staff outside the United States, contact your

local GE Healthcare office. Contact information is listed in the

back of this protocol booklet.

CONTROL DNA SEQUENCE

The control DNA included in the kit is from pUC18, a double-stranded circular

DNA of 2.7kb. A partial sequence of this DNA is given below (14).

(Universal cycle primer)

5'-G TTTTCCCAGT CACGACGTTG TA->

AACGCCAGGG TTTTCCCAGT CACGACGTTG TAAAACGACG GCCAGTGCCA

AGCTTGCATG CCTGCAGGTC GACTCTAGAG GATCCCCGGG TACCGAGCTC

60 70 80 90 100

GAATTCGTAA TCATGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT

<--CTTTAA CAATAGGCGA

110

120

130

140

150

CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG

GTGTT-5'(Reverse cycle primer)

160

GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC

210 220 230 240 250

GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA

260 270 280 290 300

ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC

310 320 330 340 350

TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT

360 370 380 390 400

CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGC

170

180

190

200

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REFERENCES

1. SANGER, F., NIKLEN, S., and COULSON, A.R. (1977) Proc. Nat. Acad. Sci.

USA 74, pp 5463-5467.

2. BIGGIN, M.D., GIBSON, T.J., and HONG, G.F. (1983) Proc. Nat. Acad. Sci.

USA 80, pp 3963-3965.

3. ZAGURSKY, R.J., CONWAY, P.S., and KASHDAN, M.A. (1991)

BioTechniques 11, pp 36-38.

4. ANSORGE, W., and LABEIT, S. (1984) J. Biochem. and Biophys. Method

10, pp 237-243.

5. SHEEN, J., and SEED, B. (1988) BioTechniques 6.

6. TABOR, S., and RICHARDSON, C.C. (1987) Proc. Nat. Acad. Sci. USA 84,

pp 4767-4771.

7. TABOR, S., and RICHARDSON, C.C. (1989) J. Biol. Chem. 264, pp 6447-

6458.

8. W. M. BARNES (1992) Gene 112, pp 29-35.

9. TABOR, S., and RICHARDSON, C.C. (1995) Proc. Nat. Acad. Sci. USA, 92,

pp 6339-6343.

10.TABOR, S., and RICHARDSON, C.C. (1989) Proc. Nat. Acad. Sci. USA 86,

pp 4076-4080.

11.HUIBREGTSE, J. M., and ENGELKE, D. R. (1988) DNA and Protein

Engineering Techniques 1, pp 39-41.

12.McMAHON, G., DAVIS, E., and WOGAN, G.N. (1987) Proc. Nat. Acad. Sci.

USA 84(14), pp 4974-8.

13.CAROTHERS A.M., URLAUB G., MUCHA J., GRUNBERGER D., and

CHASIN L.A. (1989) Biotechniques May 7(5), pp 494-6, 498-9.

14.MURRAY, V. (1989) Nucleic Acids Research Nov 11 17(21), pp 8889.

15.LEVEDAKOU, E.N. , LANDEGREN, U., and HOOD, L.E. (1989)

Biotechniques May 7(5), pp 438-42.

16.LEE, J.S. (1991) DNA Cell Biol. Jan-Feb 10(1), pp 67-73.

17.BRUMMET, S. (1991) Comments 17 No. 4, pp 22-23, Unites States

Biochemical Corp., Cleveland, OH.

18.GOUGH, J.A., and MURRAY, N.E. (1983) J. Mol. Biol. 166, pp 1-19.

19.MIZUSAWA, S., NISHIMURA, S., and SEELA, F. (1986) Nucleic Acids

Research 14, pp 1319-1324.

20.PISA-WILLIAMSON, D., and FULLER, C. W. (1992) Comments 19 No. 2, pp

1, 7, United States Biochemical Corp., Cleveland, OH.

21.DENTE, L., CESARENI, G., and CORTESE, R. (1983) Nucleic Acids

Research 11, pp 1645-1655.

22.CARLSON, A., and MESSING, J. (1984) J. Biotechnology 1, pp 253.

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