IntroductionCycle sequencing using dyelabeled terminators is a rapid and convenient method for performing enzymatic extension reactions for DNA sequencing. The benefits of this method include the following:
- Single tube reactions - Less handson time required than with dye labeled primer chemistry - Same protocol for singlestranded and doublestranded DNA and PCR fragments - Less starting template needed than with noncycling protocols - Easier sequencing of large constructs (up to 48 kb) compared to noncycling protocols - Fewer nonspecific stop peaksThe original Taq DyeDeoxy Terminator Cycle Sequencing Kit uses the thermal stable enzyme, AmpliTaq DNA Polymerase. This enzyme works well, but requires very high concentrations of the dyelabeled terminators since it discriminates strongly against incorporation of dideoxynucleotides. For this reason the completed reactions contain a large quantity of unincorporated dyelabeled terminators as well as labeled extension products. The removal of these unincorporated dye labeled terminators requires the use of a spin column or other fairly rigorous method.
In this kit AmpliTaq DNA Polymerase has been replaced with a new enzyme, AmpliTaq DNA Polymerase, FS. AmpliTaq DNA Polymerase, FS was developed specifically for fluorescent cycle sequencing with both dyelabeled primers and terminators. This enzyme is a mutant form of Taq DNA polymerase which has essentially no 5'~3'nuclease activity and has drastically reduced discrimination for dideoxynucleotides. With dye terminators this permits the use of much lower levels of dye labeled terminators in the reaction and greatly simplifies the removal of unincorporated terminators.
The ABI PRISM1M Dye Terminator Cycle Sequencing Ready Reaction kits with AmpliTaq DNA Polymerase, FS combine the unique properties of this enzyme for dye terminator sequencing with the convenience of the Ready Reaction format. In this kit the same formulation of reagents that are in the Core kit are premixed into a singletube and are ready to use. The reagents are suitable for performing fluorescence based cycle sequencing reactions on single or doublestranded DNA templates, or on PCR fragments.
The concentrations of the dyelabeled dideoxynucleotides and the deoxynucleotides in the Ready Reaction Premix have been optimized to give a balanced distribution of signal between base 10 and base 700+. The dNTP mix includes dITP in place of dGTP to minimize band compressions.
This kit was developed specifically for the preparation of samples for sequence analysis on the ABI 373A DNA Sequencer, the ABI PRisM 377 DNA Sequencer and the ABI PRISM 310 Genetic Analyzer. The reagents work well with single and doublestrand templates as wel l as PCR products.
This manual contains protocols for sequencing M13 templates, plasmids, and symmetric PCR products using PerkinElmer DNA thermal cyclers. Reactions can also be carried out on the CATALYST™ 800 Molecular Biology LabStation. General instruction are given for the use of these reagents to generate samples for the instruments mentioned here. For more detailed instructions, refer to the appropriate instrument user's manual.
Abbreviations Used in this Protocol dH2O deionized or distilled water dNTP deoxynucleotide triphosphate dITP deoxyinosine triphosphate EDTA ethylenediaminetetraacetic acid TE TrisEDTA buffer (10 mM Tris, 1 mM EDTA; pH 8.0)
MATERIALS: Kit Reagents:
This kit contains aufficient reagents to sequence 24, 100, 1000, or 5000 templates. Enough standard primer and doublestranded template for 12 control reactions is included in the 21 and 100reaction kits. The 1000 and 5000 reaction kits contain enough standard primer and template for at least 50 reactions.
Terminator Premix ADye Terminator, CDye Terminator, GDye Terminator, TDye Terminator, dITP, dATP, dCTP, dTTP, TrisHCl (pH 9.0), MgC12, thermal stable pyrophosphatase, and AmpliTaq DNA Polymerase, FS pGEM~3Zf(+) doublestranded DNA Control Template, 0.2 ~g/pL 21 M13 Primer (forward), 0.8 pmol/,uL
Reagents Supplied by the User Distilled or deionized water 25 mM EDTA (pH 8.0) with Blue Dextran, 50 mg/ml (P/N 402055) or 50 mM EDTA (pH 8.0) Deionized formamide Light mineral oil
The mineral oil is not needed if you use the CATALYST 800 Molecular Biology LabStation to prepare the sequencing reactions, or if the reactions are done in the PerkinElmer GeneAmp PCR System 9600 or 2400. 95% ethanol 70% ethanol 3 M Sodium acetate (pH 4.6) Equipment Supplied by the User 1 mL spin column (optional) We recommend CentriSep~ columns from Princeton Separations, Adelphia, NJ. Sterile pipette tips 0.6 mL doublesnap cap microcentrifuge tubes or 0.2 mL GeneAmp tubes Adjustable pipette Vortex mixer Microcentrifuge (Variablespeed for CentriSep columns) Vacuum centrifuge Thermal cycler
Storage and Use of the Kit Store the kit at 20 C. Prior to each use of the kit, allow the frozen premix stock to thaw at room temperature. Mix gently and centrifuge briefly to collect all the liquid at the bottom of each tube. Whenever possible, materials should be kept on ice during use. All solutions containing the dye terminators should be protected from light as much as is practical.
Preparation of Templates Control Templates Always perform a control reaction using a standard template. The recommend M13mpl8 for a singlestranded control, pGEM3Zf(+) for a doublestranded control.
Singlestranded DNA Templates A protocol for preparing M13 templates is provided in the ABI 3 73 DNA Sequencing System User's Manual (P/N 903204) and the ABI P7USM DNA Sequencing Guide (P/N 903563). Prepare enough template to be able to check its purity and to quantitate it accurately. The recommended concentration and quantities are shown below.
Double-stranded DNA Templates The quality of your sequencing results is directly affected by the quality of your starting DNA. The optimal procedure for preparing the plasmid depends on your particular bacterial strain and the yield of your construct. Good sequencing data has been obtained from plasmids isolated by cesiumbanding methods, a modified "mini" alkalinelysis/PEG precipitation procedure (User Bulletin Number 18), or Qiagen DNA isolation procedures. The recommended template concentrations and quantities are shown below.
Symmetric PCR Templates When sequencing symmetric templates, cycle sequencing was found to provide the most reproducible results. Although symmetric PCR fragments can be difficult to denature with traditional sequencing methods, cycle sequencing provides several chances to denature and extend the template which, in turn, insures adequate signal in the sequencing reaction.
PCR fragments need to be purlfied before sequencing. In general, any method that removes dNTPs and primers should work. We recommend Centricon~100 MicroConcentrators from Amicon (PerkinElmer P/N N9302119). The protocol for using these columns is provided under "Purifying PCR Fragments" below. If possible, quantitate the amount of purlfied PCR product by measuring the absorbance at 260 nm or some other method.
Purifying PCR Fragments Although there are many different approaches to purifying PCR fragments, Centricon100 Concentrator columns (PerkinElmer P/N N9302119). To purify PCR fragments with Centricon-100 columns 1. Assemble the Centricon100 column according to the manufacturer's recommendations. 2. Load 2 mL dH2O onto the column. 3. Add your entire sample to the column. 4. Spin the column at 3000 x g in a centrifuge with a fixedangel rotor for ten minutes
Note The manufacturer recommends a maximum speed of 1000 x g but 3000 x g works well in ABD labs. If you are following the manufacturer's guidelines, increase the amount of time of centrifugation to compensate. If the PCR primers are not completely removed with one wash, repeat this procedure starting at Step2. 5. Remove the waste receptacle and attach the collection vial. 6. Invert the column and spin it at 270 x g for two minutes to collect your sample. This should yield approximately 4060 ~L of sample. 7. Dilute the sample up to 100 pL with dH2O.
Table 1. Recommended Concentrations and Quantities of DNA
DNA Concentration (ng/pL) Quantity (ng)
Singlestranded DNA 50 100 50 100
Doublestranded DNA 100 500 250 500
PCR DNA 10 30 30 90
Protocol for Cycle Sequencing When used in conjunction with the preparation techniques described in the section "Preparation of Templates" on page 4, this protocol yields accurate sequence data for singlestranded and doublestranded DNA templates and symmetric PCR fragments to approximately 400 bases for a 24 cm well to read gel (ABI 373). Accurate sequence data beyond this range may be obtained as well. For a 34 cm welltoread gel (ABI 373), accurate sequence data for singlestranded and doublestranded templates can be obtained to approximately 500 bases. With the 48 cm well to read gel, singlestranded and doublestranded templates often yield approximately 650 bases of good data. The 36 cm welltoread gel on the ABI PRISM 377 gives approximately 600650 bases of good data.
Mixing the Reagents The type of tube required for these reactions will depend on the type of thermal cycler used. For the DNA Thermal Cycler(TCl) and the DNA Thermal Cycler 480 use 0.6 mL doublesnapcap microcentrifuge tubes. For the GeneAmp 9600 and the GeneAmp 2400 use 0.2 mL GeneAmp tubes. 1.For each reaction, mix the following reagents in a labeled tube of the appropriate size: Reagent Quantity Terminator Ready Reaction Mix 8.0 uL Template singlestranded DNA, 0.1 ,ug/ uL 0.5 1.0 uL doublestranded DNA, 0.2 ug / uL 1.5 2.5 uL PCR product, 1030 ng/ uL 3 6 uL Primer 3.2 pmole H2O q.s. Final Reaction Volume 20 uL 2. If using the DNA Thermal Cycler (TC1) or the DNA Thermal Cycler Model 480, overlay the reaction mixture with one drop of light mineral oil (approximately 40 uL). If using the CATALYST 800 Molecular Biology LabStation, refer to Section 3 in the user's manual for the reaction setup. Cycle Sequencing on the GeneAmp PCR Systems 9600 and 2400
Place the tubes in the thermal cycler, begin thermal cycling as follows: Rapid thermal ramp to 96 C 96 C for 10 seconds Rapid thermal ramp to 50 C 50 C for 5 second Rapid thermal ramp to 60 C 60 C for 4 minutes 2. Repeat Step 1 for 25 cycles. 3. Rapid thermal ramp to 4 C and hold. 4. Proceed with "Purifying Extension Products".
Note Condensation is sometimes observed on the walls of the tubes at the end of the reaction. If this is the case, it is recommended that the reaction mixture be centrifuged prior to removal of the unincorporated dye terminators.
Cycle Sequencing on the DNA Thermal Cycler (TC1 ) and the DNA Thermal Cycler Model 480 1. Place the tubes in a thermal cycler, and begin thermal cycling as follows: Rapid thermal ramp to 96 C 96 C for 30 seconds Rapid thermal ramp to 50 C 50 C for 15 seconds Rapid thermal ramp to 60 C 60 C for 4 minutes 2. Repeat Step 1 for 25 cycles. 3. Rapid thermal ramp to 4 C and hold. Proceed with "Purifying Extension Products"
Purifying Extension Products Because the amount of the dye terminators has been sign)ficantly reduced in this kit, excess terminators now can be removed by either ethanol precipitation or with spin columns. Although traces of unincorporated terminators may be seen occasionally at the front of the sequence data, this is generally minimal. Some loss in the recovery of the smallest fragments may also be observed.
To effectively remove the terminators by ethanol precipitation, pay careful attention to recommended volumes and reagent concentrations. Two protocols for ethanol precipitation are given below. The first protocol is the most reliable and gives the best results, but requires a 70% ethanol wash. The second protocol is simpler, but will result in the carryover of some unincorporated dye terminators. If desired, spin columns can also be used. CentriSep spin columns have given the most consistent results for this application.
Ethanol Precipitation Protocol 1 1. For each reaction, prepare a 1.5 mL microcentrifuge tube by adding the following: 2.0 ,uL 3 M Sodium acetate, pH 4.6 50 ,uL 95% ethanol Note : 3 M sodium acetate, pH 5.2,and 3 M potassium acetate, pH 5.6 appear to work equally well. 2. Transfer the entire 20,uL contents of the reaction tubes to the microcentrifuge tubes containing the ethanol solution. Vortex and place on ice for 10 minutes. CATALYST 2.02 users may add ethanol directly to the product tubes. 3. Centrifuge in a microcentrifuge at maximum speed for 1530 minutes. 4.Carefully aspirate the ethanol solution with a micropipetter. Remove as completely as possible. Rinse the pellet by adding 250 ,uL 70% ethanol. At this point it is not necessary to centrifuge. 5. Carefully aspirate all the alcohol solution with a micropipetter. Use a KimWipe to remove any alcohol from the sides of the tube. Be careful not to disturb the pellet, which may or may not be visible. 6. Dry the pellet in a vacuum centrifuge.
Ethanol Precipitation Protocol 2 1. For each reaction prepare a 1.5 mL microcentrifuge tube containing 35 uL 95% alcohol. 2. Transfer all 20 pL of each reaction to the tubes. Mix the contents by vortexing, and incubate on ice for 10 minutes. CATAKYST 2.02 users may add 35 of 95% alcohol directly to the product tubes. 3. Centrifuge in a microcentrifuge at maximum speed for 1530 minutes. 4. Carefully aspirate all of the liquid from the pellet. If any alcohol remains on the sides of the tube, wipe carefully with a KimWipe. Be careful not to disturb the pellet, which may or may not be visible. 5. Dry the pellet in a vacuum centrifuge.
Spin-Column Purification CentriSep spin columns can be used with either a variablespeed microcentrifuge or in preparative and clinical centrifuges. Details for the use of these columns can be found in the instructions supplied by the manufacturer.
The entire spincolumn procedure should be performed without interruption to ensure optimal results. It is especially important that you do not allow the column to dry excessively. Do not process more columns than you can conveniently handle at one time.
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