Isolation of Specific DNA sequences
- In order to prove the properties of DNA in relation to specific functions, special classes or segments of DNA must be separated from host.
- Many techniques are available:
- The separation of rapidly annealing portion and slowly annealing unique segments of mammalian DNA is accomplished on nitrocellulose filters. The highly repetitive sequences involve repetition of a short basic unit.
- The molecule ethidium can be used for separating small plasmids from the bacterial chromosomes. The molecule ethidium can insert itself between bases of a DNA molecule. If the molecule is sufficiently flexible to untwist a bit provides the space for ethidium to squeeze in. Hence, when a DNA is isolated from bacterial or eukaryotic cells, the large linear molecules binds much more ethidium than small circular molecules which thus remain denser.
- Another method is using the ability of some proteins to recognize such sequences. For eg: Enzyme RNA polymerase responsible for transcription must initiate the process of attachment to specific regions at the beginning of genes. After preparation of DNA is exposed to RNA polymerase, the mixture can be digested with a nuclease that breaks down the DNA chains. Those sequence to which enzyme is bound are physically sheltered from the nucleolytic action so they can be recovered intact after the nuclease treatment.
- An important tool for isolation of specific DNA sequences resulted from a study of tumor causing viruses.