Cycle Sequencing: Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers

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1 1 of 6 4/29/2009 2:48 PM Cite as: Cold Spring Harb. Protoc.; 2006; doi: /pdb.prot3791 Protocol Cycle Sequencing: Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers Joseph Sambrook and David W. Russell This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001 INTRODUCTION In this protocol, asymmetric PCR is used to generate single-stranded DNA templates for dideoxy-sequencing. MATERIALS 5x Cycle-sequencing buffer AmpliTaq CS or other exonuclease-deficient versions of Taq DNA polymerase (5 units/µl) EDTA (0.05 M, ph 8.0) Formamide loading buffer Oligonucleotide Primers, radiolabeled at the 5' terminus with 33 P or 32 P To prepare end-labeled primers, please see Phosphorylating the 5' Termini of Oligonucleotides. Template DNAs Plasmids, cosmids, bacteriophage, and bacteriophage M13 DNAs, purified from large- or small-scale cultures can be used as templates. The table below shows the amounts of each type of template required. View larger version (3K): [in this window] [in a new window] Thermostable DNA polymerase For advice on which enzyme to use, please see Table in Protocol 5 in the print version of the manual. This protocol has been written with AmpliTaq CS in mind, but it will work well for thermostable enzymes with similar properties.

2 of 6 4/29/2009 2:48 PM Tris-Cl (1 M, ph 8.0) dntp solutions of the four dntps, each at 1.0 mm ddntp extension/termination mixtures Assemble the mixtures according to the table below.

2 2 of 6 4/29/2009 2:48 PM Tris-Cl (1 M, ph 8.0) dntp solutions of the four dntps, each at 1.0 mm ddntp extension/termination mixtures Assemble the mixtures according to the table below. View larger version (3K): [in this window] [in a new window] Dispense as 50-µl aliquots; store frozen at -20 C. The ratios of ddntp:dntp in the four extension/temination mixtures are 20:1, 40:1, 30:1, and 10:1 for C, T, A, and G, respectively. These ratios were optimized for reactions catalyzed by AmpliTaq CS DNA polymerase. When using other thermostable DNA polymerases, the ratios may need to be re-optimized. ddntp solutions of the four ddntps, each at 5.0 mm METHOD 1. Transfer 4 µl of appropriate ddntp extension/termination mixture (please see table in Materials [Buffers and Solutions] section) into 0.5-ml color-coded microcentrifuge tubes or into individual wells of a heat-stable microtiter plate prelabeled C, T, A, and G (i.e., 4 µl of ddctp mixture into tube/well labeled C, 4 µl of ddttp mixture into tube/well labeled T, etc.). Store the tubes/microtiter plates on ice. 2. To the side of each tube or well add: double-stranded template DNA fmoles 1.5 pmoles of 5' 32 P-end labeled primer* 1.0 µl 5x cycle-sequencing buffer 2.0 µl H 2 O to 5.0 µl *If using a 33 P end-labeled primer: 1.5 µl of a standard preparation of 33 P-labeled oligonucleotide contains approx. 5.0 pmoles of primer. To maintain the stoichiometry of the components of the sequencing reaction, increase the amount of template DNA approx. 3-fold ( fmoles) without increasing the total volume of the reaction. 3. Dilute an aliquot of the preparation of AmpliTaq CS DNA polymerase with 1x cycle-sequencing buffer to a final concentration of unit/µl. Add 1 µl of the diluted enzyme to the side of each tube or well.

3 3 of 6 4/29/2009 2:48 PM 4. Mix the reagents by flicking the tubes with a finger or shaking the microtiter plate. If necessary, overlay each reaction with a drop of light mineral oil, cap the tubes, and centrifuge them at 2000 rpm for 2 seconds in a microcentrifuge or briefly in a centrifuge with microtiter plate adaptors. 5. Load the tubes or plate into a thermal cycler, preheated to 95'C. Then begin thermal cycling according to the program outlined below. IMPORTANT Do not delay in starting the program. Make sure that the samples are not exposed for >3 minutes to 95'C during the loading/preheating step. Otherwise, the DNA polymerase may be inactivated. Cycle Number Denaturation Annealing Polymerization Preheating 60 sec at 95'C cycles 30 sec at 95'C 30 sec at 55'C 60 sec at 72 C 10 cycles 30 sec at 95'C 60 sec at 72 C 60 sec at 72 C Times and temperatures may need to be adapted to suit the particular reaction conditions. 6. Remove the tubes or plates from the thermal cycler and add 5 µl of formamide loading buffer to each cyclesequencing reaction. 7. The reactions may be stored for up to 5 days at -20 C or analyzed directly by denaturing gel electrophoresis (Preparation of Denaturing Polyacrylamide Gelsor Preparation of Denaturing Polyacrylamide Gels Containing Formamide). After heat denaturation (2 minutes at 100 C) and quick cooling on ice, load 3 µl of each of the C, T, A, and G reactions into individual wells of a sequencing gel. REFERENCES 1. Lee, E., Nestorowicz, A., Marshall, I.D., Weir, R.C., and Dalgarno, L Direct sequence analysis of amplified dengue virus genomic RNA from cultured cells, mosquitoes and mouse brain. J. Virol. Methods 37: [Medline] 2. Murray, V Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res. 17: [Free Full Text] 3. Lee, J.S Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase. DNA Cell Biol. 10: [Medline] 4. Craxton, M "Linear amplification sequencing, a powerful method for sequencing DNA. ". Methods 3: Carothers, A.M., Urlaub, G., Mucha, J., Grunberger, D., and Chasin, L.A Point mutation analysis in a mammalian gene: Rapid preparation of total RNA, PCR amplification of cdna, and Taq sequencing by a novel method. BioTechniques 7: [Medline] Caution Formamide Formamide is teratogenic. The vapor is irritating to the eyes, skin, mucous membranes, and upper respiratory tract. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood when working with concentrated solutions of formamide. Keep working solutions covered as much as possible.

4 4 of 6 4/29/2009 2:48 PM Caution Radioactive substances Radioactive substances: When planning an experiment that involves the use of radioactivity, consider the physicochemical properties of the isotope (half-life, emission type, and energy), the chemical form of the radioactivity, its radioactive concentration (specific activity), total amount, and its chemical concentration. Order and use only as much as needed. Always wear appropriate gloves, lab coat, and safety goggles when handling radioactive material. X rays and gamma rays are electromagnetic waves of very short wavelengths either generated by technical devices or emitted by radioactive materials. They might be emitted isotropically from the source or may be focused into a beam. Their potential dangers depend on the time period of exposure, the intensity experienced, and the wavelengths used. Be aware that appropriate shielding is usually made of lead or other similar material. The thickness of the shielding is determined by the energy(s) of the X rays or gamma rays. Consult the local safety office for further guidance in the appropriate use and disposal of radioactive materials. Always monitor thoroughly after using radioisotopes. A convenient calculator to perform routine radioactivity calculations can be found at: Cycle-sequencing Buffer 200 mm Tris-Cl (ph 8.8) 25 mm MgCl 2 For a 5x buffer. EDTA To prepare EDTA at 0.5 M (ph 8.0): Add g of disodium EDTA 2H 2 O to 800 ml of H 2 O. Stir vigorously on a magnetic stirrer. Adjust the ph to 8.0 with NaOH (~20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the ph of the solution is adjusted to ~8.0 by the addition of NaOH. Formamide loading buffer 80% (w/v) deionized formamide 1 mg/ml xylene cyanol FF 1 mg/ml bromophenol blue 10 mm EDTA (ph 8.0) Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20 C.

5 5 of 6 4/29/2009 2:48 PM Tris-Cl Tris base HCl To prepare a 1 M solution, dissolve g of Tris base in 800 ml of H 2 O. Adjust the ph to the desired value by adding concentrated HCl. ph HCl ml ml ml Allow the solution to cool to room temperature before making final adjustments to the ph. Adjust the volume of the solution to 1 L with H 2 O. Dispense into aliquots and sterilize by autoclaving. If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The ph of Tris solutions is temperature-dependent and decreases ~0.03 ph units for each 1 C increase in temperature. For example, a 0.05 M solution has ph values of 9.5, 8.9, and 8.6 at 5 C, 25 C, and 37 C, respectively. dntp solution Dissolve each dntp (deoxyribonucleoside triphosphates) in H 2 O at an approximate concentration of 100 mm. Use 0.05 M Tris base and a micropipette to adjust the ph of each of the solutions to 7.0 (use ph paper to check the ph). Dilute an aliquot of the neutralized dntp appropriately, and read the optical density at the wavelengths given in the table below. Calculate the actual concentration of each dntp. Dilute the solutions with H 2 O to a final concentration of 50 mm dntp. Store each separately at -70 C in small aliquots. For polymerase chain reactions (PCRs), adjust the dntp solution to ph 8.0 with 2 N NaOH. Commercially available solutions of PCR-grade dntps require no adjustment. Base wavelength (nm) Extinction Coefficient (E) (M -1 cm -1 ) A x 10 4 G x 10 4 C x 10 3 T x 10 3 For a cuvette with a path length of 1 cm, absorbance = EM. 100 mm stock solutions of each dntp are commercially available (Pharmacia).

6 6 of 6 4/29/2009 2:48 PM ddntp solution Dissolve each ddntp (dideoxyribonucleoside triphosphates) in H 2 O at an approximate concentration of 100 mm. Use 0.05 M Tris base and a micropipette to adjust the ph of each of the solutions to 7.0 (use ph paper to check the ph). Dilute an aliquot of the neutralized ddntp appropriately, and read the optical density at the wavelengths given in the table below. Calculate the actual concentration of each ddntp. Dilute the solutions with H 2 O to a final concentration of 50 mm ddntp. Store each separately at -70 C in small aliquots. Base Wavelength (nm) Extinction Coefficient (E)(M-1cm-1) A x 10 4 G x 10 4 C x 10 3 T x 10 3 For a cuvette with a path length of 1 cm, absorbance = Em. Copyright 2006 by Cold Spring Harbor Laboratory Press. Online ISSN: Terms of Service All rights reserved. Anyone using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used with caution. For a full listing of cautions, click here. All rights reserved. No part of these pages, either text or images, may be used for any reason other than personal use. Reproduction, modification, storage in a retrieval system or retransmission, in any form or by any means-electronic, mechanical, or otherwise-for reasons other than personal use is strictly prohibited without prior written permission. CiteULike Connotea Del.icio.us Digg Reddit Technorati What's this?

Use TBE at a working strength of 1x (89 mm Tris-borate, 2 mm EDTA) for polyacrylamide gel electrophoresis.

Use TBE at a working strength of 1x (89 mm Tris-borate, 2 mm EDTA) for polyacrylamide gel electrophoresis. 1 of 6 4/29/2009 2:49 PM Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot3815 Protocol Detection of Mutations by Single-strand Conformational Polymorphism Joseph Sambrook and David W. Russell