Molecular Techniques and Methods

Cycle Sequencing of Bacterial Colonies
Containing Low-Copy-Number Plasmid DNA

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Cycle sequencing employs a Taq DNA polymerase in a temperature cycling format to perform multiple rounds of dideoxynucleotide sequencing on the template. The result of the temperature cycling is linear amplification of the sequencing product, leading to an increase in the signal generated during the sequencing reaction.

Cycle sequencing products can be labeled in a number of different ways. If radioactive labeling is used, either incorporation of an X-labeled dNTP or end-labeled primers can be used. 32P-dNTP and 35S-dNTP are the most common radiolabels used in sequencing. 32P-dNTP has the advantage of stronger signal strength but 35S has the advantage of sharper bands and a longer half-life. One disadvantage to the use of 35S-dNTP in cycle sequencing is the low labeling efficiency obtained using Taq DNA polymerase. For those who wish to use 35S-dNTP, a non-Thermus sp. DNA polymerase-based kit is better. Recently, an exonuclease-deficient DNA polymerase from Pyrococcus furiosus (Exo-Pfu) was shown to provide much stronger signals than Taq DNA polymerase when 35S-dATP was used as the radiolabel. 33P-dNTP was recently introduced as an alternative radioactive label for use in molecular biology. The beta-emission of this 33P-dNTP label has physical characteristics intermediate between 35S-dNTP and 32P-dNTP, making it ideal for DNA sequencing applications. It gives strong, sharp bands and allows long read lengths with shorter exposure times, often 2 hours. This isotope is used efficiently by Exo-Pfu polymerase and Taq DNA polymerase.

Cycle sequencing reaction results in several advantages:
  • The amount of template necessary for the sequencing reaction is greatly reduced when compared to dideoxy sequencing (0.2 ug vs. 2 ug).
  • Screening reactions can be performed on minimally prepared templates.
  • The high temperature at which the sequencing reactions are run allows the Taq DNA polymerase to synthesize through areas of secondary structure.
  • The multiple heat-denaturation steps allow double-stranded templates like plasmids, cosmids, lambda DNA, and PCR products to be sequenced without a separate denaturation step.




    MATERIALS AND SOLUTIONS

    10 X Taq Sequencing Buffer (1 ml)
    300 mM Tris-HCl (pH9.0) ---------------------- 300 ul of 1 M Tris-HCl
    50 mM MgCl2 ---------------------------------- 50 ul of 1 M MgCl2
    300 mM KCl ----------------------------------- 300 ul of 1 M KCl
    0.01% Gelatin ----------------------------------- 10 ul of 1% Gelatin
    Distilled H2O ------------------------------------ 340 ul


    Termination Mix-A (1 ml)
    2mM ddATP ------------------------------------- 20 ul of 100 mM ddATP
    100 uM dATP ----------------------------------- 1 ul of 100 mM dATP
    100 uM dCTP ----------------------------------- 1 ul of 100 mM dCTP
    100 uM dGTP ----------------------------------- 1 ul of 100 mM dGTP
    100 uM dTTP ----------------------------------- 1 ul of 100 mM dTTP
    Distilled H2O ------------------------------------ 976 ul


    Termination Mix-C (1 ml)
    1 mM ddCTP ------------------------------------ 10 ul of 100 mM ddCTP
    100 uM dATP ----------------------------------- 1 ul of 100 mM dATP
    100 uM dCTP ----------------------------------- 1 ul of 100 mM dCTP
    100 uM dGTP ----------------------------------- 1 ul of 100 mM dGTP
    100 uM dTTP ----------------------------------- 1 ul of 100 mM dTTP
    Distilled H2O ------------------------------------ 986 ul


    Termination Mix-G (1 ml)
    0.2mM ddGTP ----------------------------------- 2 ul of 100 mM ddGTP
    100 uM dATP ----------------------------------- 1 ul of 100 mM dATP
    100 uM dCTP ----------------------------------- 1 ul of 100 mM dCTP
    100 uM dGTP ----------------------------------- 1 ul of 100 mM dGTP
    100 uM dTTP ----------------------------------- 1 ul of 100 mM dTTP
    Distilled H2O ------------------------------------ 996 ul


    Termination Mix-T (1 ml)
    2mM ddTTP ------------------------------------- 20 ul of 100 mM ddTTP
    100 uM dATP ----------------------------------- 1 ul of 100 mM dATP
    100 uM dCTP ----------------------------------- 1 ul of 100 mM dCTP
    100 uM dGTP ----------------------------------- 1 ul of 100 mM dGTP
    100 uM dTTP ----------------------------------- 1 ul of 100 mM dTTP
    Distilled H2O ------------------------------------ 976 ul


    Stop Solution (1 ml)
    10 mM NaOH ------------------------------------ 2 ul of 5 M NaOH
    95% Formamide ---------------------------------- 950 ul of 100% Formamide
    20 mM EDTA ------------------------------------ 40 ul of 0.5 M EDTA
    0.05% Bromo Phenol Blue ------------------------ 10 ul of 5% Bromo Phenol Blue
    0.05% Xylene cyanol ------------------------------ 10 ul of 5% Xylene cyanol




    PROCEDURES

    Preparation of Bacterial Colonies Containing Low-Copy-Number Plasmid for Sequencing Reaction

  • The purity of the template will affect the quality of the sequence data, particularly when automated sequencing protocols are employed.

    1. Prepare a 1-ml overnight liquid culture.

    2. Collect 100-200 ul of the cells by centrifugation for 1 minute in a microfuge.

    3. Resuspend the cell pellet in 25 ul of TE.

    4. Boil the cells for 5 minutes.

    5. Vortex well and cool on ice. Then centrifuge for 1 minute in a microfuge to remove cell debris.

    6. Use 10 ul of the supernatant as the template in subsequent sequencing reactions.

  • Although the use of plasmid DNA obtained by boiling cells in this manner will not yield high-quality sequence data (only 100-200 bases of readable sequence), the technique is particularly useful when screening large numbers of site-specific mutagenesis products or screening cloning products.


    Radioactive Labeling of Sequencing Primers

    7. Prepare a Master Labeling Mix containing the following components.

    Distilled H2O
    2.5 ul
    10 x T4 Polynucelotide Kinase Buffer
    2 ul
    [r-33P]ATP or [r-32P]ATP
    (10 uCi/ ul; 1,000-5,000 uCi/ mmole)
    2 ul
    T4 Polynucleotide Kinase
    10-20 U/ 2 ul
    Final Volume
    20 ul


    8. Label 2-5 pmoles of sequencing primer for each sequencing reaction as follow.

    Master Labeling Mix from step 2
    4 ul
    Sequencing Primer (2-5 pmoles/ 10-25 ng of a 17-mer)
    1 ul


    9. Incubate for 15 minutes at 37oC.

    10. Heat-kill the T4 Polynucleotide Kinase for 10 minutes at 80oC.

    11. The removal of unincorporated [r-32P]ATP, although not critical, is recommended.
  • This can be accomplished by gel filtration through a Biospin-10 column (Bio-Rad Laboratories).
  • The labeled primer can be stored at -70oC for at least 2 weeks.


    Cycle Sequencing Reaction

    12. Prepare the Preraction Mix as follow.

    Labeled Sequencing Primer
    5 ul
    10 x Taq Sequencing Buffer
    4.5 ul
    15-50 fmole Template DNA 30-100 ng of a 3-kb plasmid
    Taq DNA Polymerase
    1-2.5 U/ 0.5 ul
    Add distilled H2O to make a final volume of
    36 ul


    13. Place tube in wet ice.

    14. Prepare one set of four sequencing reaction tubes.
    Add 2 ul each of Termination Mix.

    15. Pipette 8 ul of the Preraction Mix from step 7.

    16. Overlay 20 ul of mineral oil.

    17. Briefly centrifuge the tubes.

    18. Place the tubes in wet ice.

    19. Do PCR reaction as follow.

    1 cycle
    95oC
    1 min
    20 cycles
    95oC
    30 sec
    55oC (or, Tm-5oC)
    30 sec
    70oC
    60 sec
    10 cycles
    95oC
    30 sec
    70oC
    60 sec

  • Tm (oC) = [2 x (A+T)] + [4 x (G+C)]


    20. Stop reaction by adding 3 ul of Stop Solution.

    21. Heat at 90oC for 5 min.

    22. Load 2 ul each on a sequencing gel (6-8% denaturing-PAGE).

  • In 6% PAGE, bromo phenol blue moves like 26 nucleotides and xylene cyanol moves like 106 nucleotides.
  • In 8% PAGE, bromo phenol blue moves like 20 nucleotides and xylene cyanol moves like 80 nucleotides.




    NOTES

  • Procedures for performing fluorescence-based dideoxy sequencing reactions are available from Applied Biosystems, Inc. and rely on four dye-labeled dideoxynucleotides, which are combined in a single tube. This cycle sequencing protocol requires automatic sequencer for running and analysis of the sequencing gel. For manual nonradioactive detection, hapten-labeled primers can be used and detection is accomplished enzymatically. Most oligonucleotide synthesis suppliers can prepare these modified nucleotides. For those who synthesize their own oligonucleotides, the appropriate modified phosphoramidites can be purchased from most major suppliers. If a biotinylated oligonucleotide is used as the sequencing primer, the sequencing products are transferred to a nylon or nitrocellulose membrane by capillary action or electroblotting. After washing and blocking the membrane, a streptavidin-alkaline phosphatase (or horseradish peroxidase) conjugate is applied. This enzyme then generates either chemilumineseent or colored products from specialized substrates. Alternatively, other haptens such as fluorescein may be added to the end of the primer during synthesis. The fluorescein molecules are detected with an anti-fluorescein antibody conjugated to alkaline phosphatase (or horseradish peroxidase) and detected with appropriate substrates.


  • If it is necessary to read a sequence close to the primer, decreasing the ratio of dNTP/ ddNTP is helpful. To increase the intensity of bands further from the primer, it is suggested that the ratio of dNTP/ ddNTP be increased by a factor of 2-5.


    Problems
    Possible Reasons
    Light or Blank Film (1) The primer is not annealing efficiently. Reduce the annealing temperature or redesign the primer.
    (2) Not enough template DNA was used.
    (3) No radioactivity was added, or the radiolabel is old.
    (4) One of the reaction components is missing, or the reaction components were not thoroughly mixed.
    (5) The template DNA is contaminated. Make sure excess salt and EDTA have not contaminated the preparation.
    High Background There is too much template DNA. This can be a serious problem with short PCR products. Titrate down the amount of DNA added to the reaction.
    Bands in Multiple Lanes (1) The primer annealed at multiple sites. A higher annealing temperature may help.
    (2) Multiple templates are in the sequencing reaction. PCR products may be a particular problem if multiple products or primer-dimer artifacts are present and one of the PCR primers is used as a sequencing primer. Gel purification should alleviate the problem.
    (3) Gel compression artifacts are present. Increase the gel temperature (up to 60oC) or add formamide to the gel.
    (4) Template DNA is contaminated.
    Blurry or Smeared Bands (1) Old or improperly prepared acrylamide solutions were used.
    (2) The samples were not fully denatured prior to gel electrophoresis.
    Little or No Sample Volume
    Remaining after Cycling
    There was sample evaporation, possibly due to insufficient oil overlay.
    Add at least 25 ul of silicone or mineral oil and briefly centrifuge before cycling.


    KIT INFORMATION



    REFERENCES

  • Kretz, K, Callen, W, Hedden, V (1994) Cycle sequencing. PCR Methods Appl. 5: S107-S112.

  • Krishnan, B.H., Blakesley, RW, Berg, DE (1991) Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies. Nucleic Acids Res. 19: 1153.

  • Murray, V (1980) Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res. 17: 8880.

  • Roberts, SS (1992) Thermostable DNA polymerases heat up DNA sequencing. J. NIH Res. 4: 89-94.

  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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