Locating DNA sequences on chromosomes

• The base sequences of a purified nucleic acid can be used to locate those sequences of DNA within a chromosome that are complementary to them.
• DNA is duplicated several hundred fold in the polytene chromosome.
• Specific nucleic acids have many potential pairing partners at a complementary site to locate the genes coding for an RNA.
• The chromosome are initially squashed and fixed on the slides.
• Then the DNA is denatured with a mild alkali treatment that breaks the hydrogen bonds without liberating the DNA from the chromosome.
• The single strands are prevented from renaturing by treatment with formamide which combines with free amino groups and inhibits duplex reformation.
• Now, radioactive RNA is incubated on the chromosomes.
• After sufficient time for hybridization, the excess label is washed off.
• Single stranded RNA is removed by treatment with ribonuclease and photographic emulsion is placed on the chromosome locate DNA-RNA hybrids in an autoradiograph.