Which of the following is/are NOT the step(s) used in separation and isolation of DNA fragments by gel electrophoresis technique:

(A) Staining with ethedium bromide
(B) Exposure to UV Radiation
(C) Cutting DNA fragments into small pieces by using restriction endonuclease
(D) Denaturation of DNA fragments

Choose the correct answer from the options given below:

(D) only

The correct answer is Option (1) → (D) only

The process of cutting DNA using restriction endonucleases results in the formation of DNA fragments. These fragments can be separated using a technique called gel electrophoresis.

Since DNA fragments are negatively charged molecules, they can be separated by applying an electric field, causing them to move towards the anode through a medium or matrix. Agarose, a natural polymer extracted from seaweeds, is commonly used as the matrix. The DNA fragments separate based on their size due to the sieving effect provided by the agarose gel. Smaller fragments move farther compared to larger fragments.

The separated DNA fragments can be visualized only after staining the DNA with a compound called ethidium bromide, followed by exposure to UV radiation. Without staining, the pure DNA fragments cannot be seen in visible light. After staining, you can observe bright orange-colored bands of DNA in an ethidium bromide stained gel exposed to UV light.

Once the DNA fragments are separated, they can be cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The purified DNA fragments obtained through elution are then used in constructing recombinant DNA by joining them with cloning vectors.

Denaturation, which involves breaking hydrogen bonds between DNA strands, is not required in gel electrophoresis. Gel electrophoresis separates DNA based on fragment size without needing to denature the DNA. Denaturation is the first step in PCR. Denaturation is required to separate the double-stranded DNA sample. It is done at 94-98 ℃ for 20-30 seconds. It breaks the hydrogen bonds present between base pairs. Denaturation leads to the formation of single strands of DNA.