Molecular Techniques and Methods

DNA-DNA Hybridization
(Southern Blotting Analysis)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

DNA fragments are denatured, transferred to a nylon membrane, and immobilized by UV cross-linking. The DNA attached to the nylon membrane is then hybridized to labeled DNA or RNA probe, and autoradiography or chemiluminescent detection is used to locate the position of any DNA complementary to the labeled probe. This technique can be used not only to locate specific sequences in cloned DNA, but also to identify sequences within digests of total eukaryotic genomic DNA.




MATERIALS AND SOLUTIONS

Acid Depurination Solution (1 liter)
0.25 M HCl ---------------------------------- 21.5 ml of 11.6 M HCl
Distilled H2O --------------------------------- 979.5 ml


Denaturation Solution (1 liter)
1.5 M NaCl ---------------------------------- 300 ml of 5M NaCl
0.5 M NaOH -------------------------------- 100 ml of 5M NaOH
Distilled H2O --------------------------------- 600 ml


Neutralization Solution (1 liter)
1 M Tris-HCl (pH 8.0) ----------------------- 500 ml of 2 M Tris -HCl
1.5 M NaCl ---------------------------------- 300 ml of 5 M NaCl
Distilled H2O --------------------------------- 200 ml


Prehybridization Solution

Stock
10 ml
20 ml
20 x SSC
2.5 ml
5 ml
20% SDS
0.2 ml
0.4 ml
100 x Denhardt's Solution
0.5 ml
l ml
Salmon Sperm DNA (10 mg/ml)
0.1 ml
0.2 ml
0.5 M EDTA (pH8.0)
0.2 ml
0.4 ml
Distilled H2O
6.5 ml
13 ml

  • Denature salmon sperm DNA for 5 minutes in boiling water bath before adding to the Prehybridization Solution.
  • If using Blocking reagent, use 1% Blocking reagent.
  • If RNA blotting, use 50% formamide and add 2% Blocking reagent.


    Hybridization Solution

    Stock
    10 ml
    20 ml
    20 x SSC
    2.5 ml
    5 ml
    20% SDS
    0.2 ml
    0.4 ml
    100 x Denhardt's Solution
    0.5 ml
    l ml
    Salmon Sperm DNA (10 mg/ml)
    0.1 ml
    0.2 ml
    0.5 M EDTA
    0.2 ml
    0.4 ml
    Labeled, denatured Probe
    0.1 ml
    0.2 ml
    Distilled H2O
    5.85 ml
    11.7 ml

  • Denature labeled probe and salmon sperm DNA for 5 minutes in boiling water bath before adding to the Hybridization Solution.
  • If using Blocking reagent, use 1% Blocking reagent.
  • If RNA blotting, use 50% formamide and add 2% Blocking reagent.




    PROCEDURES

    DNA Blotting to Nylon Membrane

    1. After electrophoresis is completed, photograph the gel stained with ethidium bromide.

    DNA Source
    Amounts of DNA Needed for Hybridization/ Detection
    Bacteriophage DNA
    0.5-1 ug
    Plasmid DNA
    0.1-0.2 ug
    Genomic DNA
    10-20 ug


    2. Transfer the gel to a glass dish and trim away any unused areas of the gel with a razor blade.

    3. For genomic DNA hybridization, hydrolyze the DNA partially by acid depurination prior to alkali denaturation.
  • For acid depurination, soak the gel for 10-15 minutes in Acid Depurination Solution at room temperature.
  • For plasmid DNA (or bacteriophage DNA) hybridization, acid depurination is not necessary.

    4. Denature the DNA by soaking the gel in several volumes of Denaturation Solution for 1 hour at room temperature with constant shaking.

    5. Neutralize the gel by soaking in several volumes of Neutralization Solution for 1 hour at room temperature with constant shaking.

    6. Wrap a piece of Whatman 3MM paper around a piece of plexiglass plates. Place the wrapped support inside a large baking dish. The support should be longer and wider than the gel.
  • Fill the dish with 10 x SSC almost to the top of the support and smooth out all air bubbles in the 3MM paper with a glass rod.

    7. Invert the gel so that its original underside is now uppermost.
  • Place the gel on the damp 3MM paper. Make sure there are no air bubbles between the 3MM paper and the gel.

    8. Cut a piece of nylon membrane about 2 mm larger than the gel in both dimensions.
  • Use gloves to handle the nylon membrane.

    9. Wet the nylon membrane in a solution of 2 x SSC for 2-3 minutes.

    10. Place the wet nylon membrane on top of the gel, so that one edge extends just over the line of slots at the top of the gel.
  • Be careful to remove all air bubbles that are trapped between the gel and the membrane.

    11. Wet two pieces of Whatman 3MM paper, cut to the same size as the gel, in 2 x SSC and place them on top of the nylon membrane. Again remove all air bubbles.

    12. Cut a stack of paper towels (5 cm high) just smaller than the 3MM paper. Place the towels on the 3MM paper. Put a glass plate on top of the stack and weigh it down with a 100 g weight.
  • The objective is to set up a flow of liquid from the reservoir through the gel and the nylon membrane, so that DNA fragments are eluted from the gel and are deposited onto the nylon membrane.

    13. Allow transfer of DNA to proceed for several hours to overnight.
  • The rate of transfer of DNA depends on the size of the DNA fragment and the porosity of the gel. Small fragments of DNA (< 1 kb) transfer from an 0.8% Agarose gel within an hour or 2 while transfer of DNA greater than 15 kb takes 15 hours or longer.

    14. Remove the towels and the 3MM filters above the gel.
  • Turn over the dehydrated gel and nylon membrane and lay them, gel side up, on a dry sheet of 3MM paper.

    15. Mark the positions of the gel slots on the filter with a ball-point pen.
  • Peel off and discard the gel.

    16. Air-dry the nylon membrane.

    17. UV cross-link the DNA to the nylon membrane.


    Southern Hybridization (DNA-DNA Hybridization)

    18. Wet the membrane in 6 x SSC for 2 minutes.

    19. Slip the wet nylon membrane into a heat-sealable plastic bag.

    20. Add Prehybridization Solution to the bag (0.2 ml/cm2 of nylon membrane).

    21. Squeeze as much air as possible from the bag.
  • Seal the open end of the bag with the heat sealer.

    22. Incubate the bag for 1-2 hours submerged in a water bath at 65oC.

    23. Remove the bag from the water bath.
  • Open the bag by cutting off one corner with scissors.
  • Squeeze out as much Prehybridization Solution as possible.

    24. Add the Hybridization Solution including labeled probe to the bag.
  • Use just enough Hybridization Solution to keep the nylon membrane wet (50 ul/cm2 of nylon membrane).

    25. Squeeze as much air as possible from the bag.
  • Seal the cut edge with the heat sealer so that as few air bubbles as possible are trapped in the bag.

    26. Incubate the bag submerged in a water bath at 65oC for 12-48 hours.

    27. Remove the bag from the water bath and quickly cut along the length of three sides.
  • Remove the nylon membrane and immediately submerge it in a tray containing a solution of 2 x SSC and 0.5% SDS at room temperature.
  • Do not allow the membrane to dry out at any stage during the washing procedure.

    27. After 5 minutes, transfer the membrane to a fresh tray containing a solution of 2 x SSC and 0. 1% SDS and incubate for 15 minutes at room temperature with gentle shaking.

    28. Wash the membrane with a solution of 0.1 x SSC and 0.5% SDS.
  • Incubate at 65oC for 1-2 hours with gentle shaking.

    29. Wrap the membrane in Saran Wrap and apply to X-ray film to obtain an autoradiographic image, if radioactive probe is used.
  • If nonradioactive probe is used, continue to chemiluminescent detection.



    NOTES

  • Marker DNAs that will hybridize to the labeled probe can serve simultaneously to orient the membrane and to provide size markers directly on the autoradiograph.

  • Hybridization may also be carried out in buffers containing formamide. Each increase of 1% in the formamide concentration lowers the Tm of a DNA duplex by 0.7oC.

  • If the homology between the probe and the DNA bound to the membrane is inexact, the washing should be carried out under less stringent conditions. In general, washing should be carried out at Tm = -12oC

  • Tm = 69.3 + {0.41 X (G + C)%}
    The Tm of a duplex DNA decreases by 1oC with every increase of 1% in the number of mismatched base pairs.


    KIT INFORMATION




    REFERENCES

  • Southern E (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503.

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

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