Hybridization analysis is based on the principle that a single-stranded DNA molecule of a specific sequence (i.e., a commonly labeled "probe") can form a base pair with another immobilized DNA molecule or RNA molecule of a complementary sequence (the "target" sequence), and the stability of the hybrid molecule depends on the extent to which base-pairing occurs, making the technique useful for detecting single-copy genes in complex genomes. This technique allows the detection of single-copy genes in complex genomes.

Operation method

radiolabeling

Principle

This protocol is suitable for hybridization analysis of Southern transfers, spots and narrow line blots with radiolabeled DNA probes of 100 to 1 000 bp length.

Materials and Instruments

DNA
SDS SSC
Water Bath Incubator

Move

1. Wet the membrane with immobilized DNA with 6 x SSC.2. Place the membrane DNA side up in a hybridization tube, add ATP solution in the amount of about 1 ^{ml/cm2 membrane} , and roll it in the hybridization oven at 68°C for 3 h.3. Towards the end of the prehybridization, denature the DNA probe at 100°C for 10 min in ice.

4. Pour out the APH solution in the hybridization tube and replace it with an equal volume of preheated APH solution (68°C), add the denatured probe and hybridize overnight at 68°C under rolling.5. Pour off the APH solution, add an equal volume of 2×SSC/0.1% SDS and incubate for 10 min at room temperature on a rolling scale. 5 min later, replace the membrane wash solution.6. Replace the above wash solution with 0.2 x SSC/0.1% SDS of Hovenia et al. and incubate at room temperature for 10 min on a rolling basis before replacing the wash solution.

7. If necessary, perform two washes at 42℃ for 15 min at medium tightness with 0.2×SSC/0.1% SDS.

8. If necessary, perform high stringency washes twice at 68℃ for 15 min with 0.1×SSC/0.1% SDS.

9. Pour off the final wash solution, rinse the membrane with 2×SSC at room temperature, and absorb the excess liquid, wrap with plastic film and then radiate autoradiography.

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