Molecular Techniques and Methods

Dideoxy Chain-Termination Sequencing of Plasmid DNA

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The 2',3'-dideoxynucleotides (ddNTP) inhibit chain growth because they cannot form phosphodiester bonds. If a sequencing primer and template, mixed with the Klenow fragment, are incubated with the four essential dNTPs plus one ddNTP, a family of fragments will be generated. Each of these will have identical 5'-end and each will terminate at some specific base, depending on the ddNTP added.

Suppose that ddCTP was added. Chain growth will continue until a C is required by the template sequence. At that point the DNA polymerase may add a ddCTP, in which case chain growth will be terminated. Or, the DNA polymerase (e.g. Sequenase) might add a dCTP (since both dCTP and ddCTP are present), and chain growth will continue in the 5' to 3' direction at least until the next C is called for the template. At that point, the possibilities of chain termination or elongation again present themselves. A dideoxy sequence study requires that four separate incubations be run in parallel. They must be identical in composition except for the difference in ddNTP added. The four product mixtures are denatured with urea, and the variously terminated products are simultaneously examined on a single polyacrylamide gel. After autopradiography the sequence can be read on the X-ray film.




MATERIALS AND SOLUTIONS

10 x Denaturation Solution (1 ml)
2 M NaOH ---------------------------------- 400 ul of 5 M NaOH
2 mM Na2-EDTA --------------------------- 4 ul of 0.5 M Na2-EDTA
Distilled H2O -------------------------------- 596 ul


5 x Sequenase Buffer (1 ml)
200 mM Tris-HCl (pH 7.5) ------------------ 200 ul of 1 M Tris-HCl
100 mM MgCl2 ----------------------------- 100 ul of 1 M MgCl2
250 mM NaCl ------------------------------- 50 ul of 5 M NaCl
Distilled H2O -------------------------------- 650 ul


5 x Labeling Mix (1 ml)
7.5 uM dGTP ------------------------------- 7.5 ul of 1 mM dGTP
7.5 uM dCTP ------------------------------- 7.5 ul of 1 mM dCTP
7.5 uM dTTP ------------------------------- 7.5 ul of 1 mM dTTP
Distilled H2O -------------------------------- 977.5 ul


ddG Termination Mix (1 ml)
80 uM dGTP -------------------------------- 8 ul of 10 mM dGTP
80 uM dATP -------------------------------- 8 ul of 10 mM dATP
80 uM dCTP -------------------------------- 8 ul of 10 mM dCTP
80 uM dTTP -------------------------------- 8 ul of 10 mM dTTP
8 uM ddGTP -------------------------------- 8 ul of 1 mM ddGTP
50 mM NaCl -------------------------------- 10 ul of 5 M NaCl
Distilled H2O -------------------------------- 950 ul


ddA Termination Mix (1 ml)
80 uM dGTP -------------------------------- 8 ul of 10 mM dGTP
80 uM dATP -------------------------------- 8 ul of 10 mM dATP
80 uM dCTP -------------------------------- 8 ul of 10 mM dCTP
80 uM dTTP -------------------------------- 8 ul of 10 mM dTTP
8 uM ddATP -------------------------------- 8 ul of 1 mM ddATP
50 mM NaCl -------------------------------- 10 ul of 5 M NaCl
Distilled H2O -------------------------------- 950 ul


ddT Termination Mix (1 ml)
80 uM dGTP -------------------------------- 8 ul of 10 mM dGTP
80 uM dATP -------------------------------- 8 ul of 10 mM dATP
80 uM dCTP -------------------------------- 8 ul of 10 mM dCTP
80 uM dTTP -------------------------------- 8 ul of 10 mM dTTP
8 uM ddTTP -------------------------------- 8 ul of 1 mM ddTTP
50 mM NaCl -------------------------------- 10 ul of 5 M NaCl
Distilled H2O -------------------------------- 950 ul


ddC Termination Mix (1 ml)
80 uM dGTP -------------------------------- 8 ul of 10 mM dGTP
80 uM dATP -------------------------------- 8 ul of 10 mM dATP
80 uM dCTP -------------------------------- 8 ul of 10 mM dCTP
80 uM dTTP -------------------------------- 8 ul of 10 mM dTTP
8 uM ddCTP -------------------------------- 8 ul of 1 mM ddCTP
50 mM NaCl -------------------------------- 10 ul of 5 M NaCl
Distilled H2O -------------------------------- 950 ul


Enzyme Dilution Buffer (1 ml)
10 mM Tris-HCl (pH 7.5) ------------------- 10 ul of 1 M Tris-HCl
5 mM DTT ---------------------------------- 5 ul of 1 M DTT
0.05% BSA --------------------------------- 50 ul of 1% BSA
Distilled H2O -------------------------------- 935 ul


Formamide Loading Buffer (1 ml)
100% Formamide --------------------------- 1ml
20 mM Na2-EDTA ------------------------- 40 ul of 0.5 M Na2-EDTA
0.05% BPB -------------------------------- 10 ul of 5% BPB
0.05% Xylene cyanol ----------------------- 10 ul of 5% Xylene cyanol




PROCEDURES

Denaturation of Template and Primer Annealing

  • The purity of the template will affect the quality of the sequence data.

    1. Purify the template DNA by using DNA isolation kit.

    2. Add 1/10th vol. of 10 x Denaturation Solution to 1-5 ug template DNA.
  • Denature DNA at 37oC for 30 min.

    3. Add 1/10th vol. of 3 M Sodium acetate (pH5.8) and 2-4 vol. of 100% ethanol.

    4. Precipitate denatured DNA at -20oC for 30 min.

    5. Centrifuge for 15 min. at 13 krpm at 4oC.
  • Remove supernatent.

    6. Spin down liquid briefly.
  • Remove any residual ethanol by Kimwipe.

    7. Air dry DNA briefly.

    8. Add the following to DNA template for sequencing reaction.

    DNA template
    1-5 ug
    Distilled H2O
    7 ul
    0.5 pmole/ul Primer
    3-5 ng/ 1 ul
    5 x Sequenase Buffer
    2 ul


    9. Anneal at 37oC for 20 min.



    Synthesis Reaction

    10. Add the following to primer-annealed DNA template.

    0.1 M DTT
    1 ul
    1 x Labeling Mix (diluted by H2O)
    2 ul
    a-35S dATP or a-33P dATP (10 uCi/ul, 1,000 Ci/mmole)
    5 uCi/ 0.5 ul


    11. Vortex and spin down briefly.

    12. Add 2 ul of 8 x diluted Sequenase (diluted by Enzyme Dilution Buffer).
  • Mix by pipetting up and down ONE time. Do NOT vortex.

    13. Incubate at room temperature for 2-5 min.



    Chain Termination Reaction.

    14. Dispense 2.5 ul of each Termination Mix in the microwell titerplate.

    15. Terminate raction by adding 3.5 ul Reaction Mix from step 13 in each well containing 2.5 ul Termination Mix.
  • Mix by pipetting up and down ONE time.

    16. Incubate for 3-5 min. at 37-43oC.

    17. Terminate the reaction by adding 4 ul of Formamide Loading Buffer.

    18. Denature sample at 90oC for 2-3 min.

    19. Load 2 ul in each well of the Sequencing Gel.

    20. Electrophoresis at 60-70 constant watts for 2-4 hours.




    NOTES

  • For sequencing GC-rich templates, dGTP should be replaced with 7-deaza-dGTP to overcome compression artifacts that are known to occur during sequencing gel electrophoresis. dGTP can also be replaced with dITP, but dITP may not be a good substrate for all DNA polymerases.

    KIT INFORMATION



    REFERENCES

  • Sanger, F, Nicklen, S, Coulson, AR (1977) DNA sequencing with chain-termination inhibitors. PNAS 74: 5463-6467.

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

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