Molecular Techniques and Methods

Chemical Sequencing of DNA
(Maxam and Gilbert's Specific-Base Cleavage Method)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This procedure is directly applicable to any single- or double-stranded DNA species of as many as 500 base pairs in length.

  • Treatment with dimethyl sulfate (DMS) and base results in ring opening of the imidazole portion of the purine (A or G). Treatment with piperidine then displaces the ring-opened A or G residue and at the same time breaks the 3'- and 5'-phosphate bonds to the associated sugar. As a result, the DNA has been cleaved into two fragments at the point of attack.
  • Strong G/ weak A (G>A) cleavage - Because G reacts 5 x fold fatser with DMS than does A, an autoradiogram has a pattern of light A and dark G bands, reflecting the difference in reaction rates between G and A.
    Strong A/ weak G (A>G) cleavage - By heating the methylated DNA in mildly acid medium, an autoradiogram has a pattern of light G and dark A bands, reflecting the difference in reaction rates between G and A.

  • Hydrazine attacks pyrimidined rings (C and T), leading to the formation of new pyrazolone structures. Continued reaction with hydrazine splits out the pyrazolone, opens the deoxyribose ring, and produces sugar hydrozone. Treatment with piperidine forms a piperidone and cleaves the 3'- and 5'-phosphate bonds, resulting in cleavage of the DNA strand. Specificity for C site cleavage can be accomplished by replacing water in the reaction mixture with 5 M NaCl. Salt addition markedly depresses reactivity of T with the hydrazine.

  • The major disadvantage of the chemical sequencing is that it takes more time to produce the same amount of sequence.
    (1) The DNA has to be end-labeled and then reisolated prior to the actual chemical sequencing reactions, a process that usually requires an additional day.
    (2) Because more DNA is used in the reaction and because the lower specific activity of the sequenced DNA requires the use of an intensifying screen in the autoradiography, bands are not as sharp as in the enzymatic method and therefore it is difficult to obtain reliable sequence past about nucleotide 250.

  • Nevertheless, the chemical method is often useful for following reasons.
    (1) It enables one to begin sequencing anywhere in the clone where a restriction site occurs without any further subcloning. The sequence thus obtained can then be used to synthesize oligonucleotide primers for enzymatic sequencing.
    (2) In cases of regions that give poor results in the enzymatic reactions (because of secondary structures that inhibit the polymerase enzyme), the chemical method almost always resolves the problem and yields the correct sequence.




    MATERIALS AND SOLUTIONS

    G Buffer (10 ml)
    50 mM Sodium cacodylate (pH. 8.0) ------------- 80 mg
    Add distilled H2O to make a final volume of ------ 10 ml
  • Adjust pH 8.0 with 0.1 M NaOH.


    CT/C Stop (10 ml)
    0.3 M Sodium acetate (pH 7.0) ------------------- 1 ml of 3 M Sodium acetate
    1 mM EDTA ------------------------------------- 20 ul of 0.5 M EDTA
    Distilled H2O ------------------------------------- 8.98 ml


    GA Stop (10 ml)
    0.3 M Sodium acetate (pH 7.0) ------------------- 1 ml of 3 M Sodium acetate
    Distilled H2O ------------------------------------- 9 ml


    G Stop (10 ml)
    1.5 M Sodium acetate (pH 7.0) ------------------- 5 ml of 3 M Sodium acetate
    1 M 2-Mercaptoethanol -------------------------- 0.7 ml of 14.3 M 2-Mercaptoethanol
    Distilled H2O ------------------------------------- 4.3 ml


    10% Formic acid (1 ml)
    Formic acid -------------------------------------- 100 ul
    Distilled H2O ------------------------------------- 900 ul


    Carrier DNA (1 ml)
    Any Plasmid DNA ------------------------------- 1 mg
    Distilled H2O ------------------------------------- 1 ml


    Carrier tRNA (1 ml)
    Any tRNA --------------------------------------- 10 mg
    Distilled H2O ------------------------------------- 1 ml


    Formamide Loading Buffer (1 ml)
    100% Formamide -------------------------------- 1 ml
    0.1 % (w/v) Bromophenol blue ------------------- 20 ul of 5% Bromophenol blue
    0.1 % (w/v) Xylene cyanol ----------------------- 20 ul of 5% Xylene cyanol




    PROCEDURES

    1. End label at one end of the DNA fragment (500 bp) to be sequenced.
  • Do not label both ends of the DNA fragment.

    2. Purify the end-labeled DNA fragment and resuspend in distilled H2O.

    3. Mark four 1.5 ml tubes and add the following solutions.

    G tube
    Carrier DNA (1 mg/ml) - 1 ul
    G Buffer - 200 ul
    Labeled DNA - 5ul
    GA tube
    Carrier DNA (1 mg/ml) - 1 ul
    Distilled H2O - 10 ul
    Labeled DNA - 10 ul
    CT tube
    Carrier DNA (1 mg/ml) - 1 ul
    Distilled H2O - 10 ul
    Labeled DNA - 10 ul
    C tube
    Carrier DNA (1 mg/ml) - 1 ul
    5 M NaCl - 15 ul
    Labeled DNA - 5ul


    4. Mark up four 1.5 ml "Stop" tubes and add the following solutions.

    G Stop
    tRNA (10 mg/ml) - 2 ul
    G Stop Solution - 50 ul
    Ethanol - 1 ml
    AG Stop
    tRNA (10 mg/ml) - 2 ul
    AG Stop Solution - 200 ul
    Ethanol - 1 ml
    CT Stop
    tRNA (10 mg/ml) - 2 ul
    CT/C Stop Solution - 200 ul
    Ethanol - 1 ml
    C Stop
    tRNA (10 mg/ml) - 2 ul
    CT/C Stop Solution - 200 ul
    Ethanol - 1 ml


    5. To start the reactions, add the following.

    Tubes from Step 3
    Reaction
    G tube
    Add 1 ul Dimethyl sulfate (DMS)
    5 min at room temperature
    AG tube
    Add 3 ul 10% Formic acid
    15 min at 37oC
    CT tube
    Add 30 ul Hydrazine (95% anhydrous)
    9 min at room temperature
    C tube
    Add 30 ul Hydrazine (95% anhydrous)
    11 min at room temperature


    6. Stop each reaction by pipetting the contents of the corresponding Stop Tube from step 4 into the Reaction Tube of step 5.

    7. Cap the reaction tubes, shake briefly but vigorously, and place in a dry ice-ethanol bath (-80oC) for 3-10 min.
  • (3 min are enough, but the tubes can be left there for up to 10 min if other reactions are not done yet; do not leave for longer than 10 min).

    8. Centrifuge at 4oC for 7 min, discard supernatant, aspirate the rest of the liquid with a drawn Pasteur pipet.

    9. Add 1 ml of 100% ethanol to the tube, invert twice, and centrifuge for 2 min.
  • Aspirate as before, and dry in a vacuum for 10 min.

    10. To each reaction tube, add 100 ul of the 10 % Piperidine solution (just prepared) (do not shake the tubes as there is no need to resuspend the DNA) and place the uncapped tubes in a 90oC heat-block.
  • After 15-30 s, cap the tubes and let stand for 30 min.

    11. Remove the tubes from the heat-block, let stand at room temperature for 2-5 min, centrifuge briefly to get the condensation to the bottom.
  • Puncture one hole in the cap with a syringe, then place in dry ice-ethanol bath for 5 min.

    12. Place the tubes in a SpeedVae machine and lyophilize for 2 hours.
  • Vacuum should be below 100 millitorr.

    13. Prior to gel electrophoresis, add 10 ul of Formamide Loading Buffer to each tube, resuspend the sample by shaking and then a brief centrifugation, and place the tubes in the 90oC heat-block for 10 min.

    14. Load 1-2 ul per sample to sequencing gel.




    NOTES

    G reaction This reaction is usually very clean, but it is the reaction most sensitive to prolonged incubation and to the quality of the reactive reagent, DMS. If reaction proceeds longer than the allotted time or if old or bad quality dimethyl sulfate is used, excessive and nonspecific degradation of DNA will occur.
    AG reaction This is usually a trouble-free reaction.
    CT and C reactions The quality of the hydrazine should be good (it does not have to be exceptional), otherwise excessive and nonspecific degradation will occur. Sometimes faint bands will be seen in the C and CT lanes when the base is G (a strong band is then observed in the G lane). The likely explanation is that the pH in the reaction tubes is too low (there is no buffer in the C and CT reaction tubes, but carryover with the DNA sample might cause this to happen). However, these faint bands are not nearly as strong as the signal in the G lane or as bona fide bands of C and T bases.

    KIT INFORMATION



    REFERENCES

  • Maxam, AM, Gilbert, W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65: 499-560.

  • Sanger, F, Nicklen, S, Coulson, AR (1977) DNA sequencing with chain-terminating inhibitors. PNAS 74: 5463-5467.

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

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