Molecular Techniques and Methods

Genome Walking by Inverse Polymerase Chain Reaction

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

IPCR (Inverse Polymerase Chain Reaction) leads to the amplification of previously unknown sequences because the primers that initially face away from each other on the linear template can be made to face each other as in normal PCR following circularization of the template. Further amplification with nested primers ensures the integrity of the final product, which can be sequenced directly.


MATERIALS




PROCEDURES

1. Prepare genomic DNA according to the procedure.
  • Genomic DNA needs to be clean enough to be readily digested by restriction enzymes and not to be inhibitory to ligation of the DNA.

    2. Dissolve the DNA in TE.

    3. Digest 1 ug DNA with a number (5-7 enzymes) of restriction enzymes in separate tubes in a total volume of 10 ul. Digest to completion.
  • In order to obtain sequence only from the upstream region, the enzymes chosen must cleave within the known sequence.
  • The length of the fragments expected from each digest can be determined by Southern blots. However, in practice, it is easier to cleave with five different enzymes in order to ensure that at least one or two of the digests yield fragments that are neither too large to be amplified (2-3 kb with Taq polymerase) nor too small to be worth the effort in sequencing the fragment.

    4. Heat-inactivate the enzyme at 68oC for 10 min if it is heat-labile or alternatively extract with phenol: chloroform and ethanol precipitate with 1/10th vol 3 M Sodium acetate (pH 5.2) and 2.5 vol ethanol if heat-stable.

    5. After precipitation, wash the DNA pellet with 70% Ethanol and resuspend to 10 ul TE.

    6. Take 2 ul (0.2 ug) of heat-inactivated or ethanol-precipitated digested DNA and setup a self-ligation reaction by adding the following components.

    Restriction Enzyme Digested DNA
    0.2 ug/ 2 ul
    10 x T4 DNA Ligase Buffer
    10 ul
    10 mM ATP
    10 ul
    T4 DNA Ligase (3 U/ ul)
    4 ul
    Add distilled H2O to make a final volume of
    100 ul


    7. Ligate 16 hours at 14oC.

    8. Remove 10 ul from each tube and add directly to a 100 ul PCR reaction containing the internal pair of the nested primers.
  • Typical PCR reaction conditions are used as follow.

    1 cycle
    Denaturation
    95oC
    60 sec
    35-40 cycles
    Denaturation
    95oC
    10 sec
    Annealing
    Tm-5oC
    60 sec
    Extension
    72oC
    3 min
    1 cycle
    Final Extension
    72oC
    5-10 min


    9. Ethanol precipitate the PCR reactions with 1/10th 3 M Sodium acetate (pH 5.2) and 2.5 vol Ethanol.

    10. Run each precipitated DNA in a single well of a 1.2% Agarose gel.
  • Visualize the bands over a long-wavelength UV-transilluminator and use a toothpick to remove a small amount of amplified product into 5 ul of distilled H2O.

    11. Use the total amount of first-round PCR product in a second round PCR reaction with nested primers.

    12. After the temperature cycling is complete, ethanol precipitate and run each product on an agarose gel.

    13. Purify the amplified DNA products using any kit.

    14. Sequence the PCR product from both directions using nested primers.




    NOTES

  • Diluting the digested DNA into a 100 ul self-ligation reaction ensures that the DNA concentration does not exceed 3 ug/ml. Experimentally, it has been found that at DNA concentrations below 3 ug/ml circles rather than linear concatamers tend to form. Also ligating in a 100 ul reaction ensures that any carried-over restriction enzyme buffer does not significantly alter the composition of the ligation reaction components.

  • Not all of the 1st round PCR reactions will work because either the product was too large to be amplified under the conditions used for PCR or the restriction enzyme digestion and/or the self-ligation steps were not efficient.

  • A much greater amount of the 2nd round PCR products should be visible following ethidium bromide staining compared to the 1st round PCR products, and their size should have dropped by using nested primers. If both of these criteria are met, it is almost certain that the product derives from a specific amplification into the previously unknown promoter region.




    KIT INFORMATION




    REFERENCES

  • Collins, FS, Weissmann, SM (1984) Directional cloning of DNA fragments at a large distance from an initial probe: A circularization method. PNAS 81: 6812-6816.

  • Mizobuchi, M, Frohman, LA (1993) Rapid amplification of genomic DNA ends. Biotechniques 15: 215-216.

  • Parker, JD, Rabinovitch, PS, Burtner, GC (1991) Targeted gene walking polymerase chain reaction. Nucl. Acids Res. 19: 3055-3060.

  • Shyamala, V, Ames, GF (1989) Genome walking by single-specific primer polymerase chain reaction: SSP-PCR. Gene 84: 1-8.

  • Siebert, PD, Chenchik, A, Kellogg, DE, Lukyanov, KA, Lukyanov, SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucl. Acids Res. 23: 1087-1088.

  • Triglia, T, Peterson, MG, Kemp, DJ (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucl. Acids Res. 16: 8186.



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