Explain the separation and isolation of DNA in DNA fingerprinting.

A very quick way to analyse the differences in the DNA sequences of two individuals is called DNA fingerprinting. It involves the identification of differences in repetitive DNA. There are many applications of DNA fingerprinting that is used as a tool in forensic investigations, for settling paternal dispute, etc. Separation and isolation of DNA is an important method in this technique. It includes the following steps: Isolation of DNA - DNA is isolated from plant or animal cells with the help of suitable enzymes. Cutting of DNA - Restriction endonucleases are used for the fragmentation of isolated DNA Amplification of DNA fragments - The DNA is amplified in multiple copies with the help of polymerase chain reaction (PCR) using enzymes, primer, free nucleotides at different temperatures. PCR includes three stages in one cycle (denaturation, annealing, extension), each cycle is repeatedly carried out in this process of amplification of DNA. Separation of DNA: The DNA fragments are separated based on their charge and size using a technique called gel electrophoresis. In this technique, DNA fragments are move towards anode through a medium under an electric field due to the negative charge of these molecules. Agarose gel is used as the medium through which DNA fragments are moved. These fragments are separated according to their size due to the sieving effect of the gel. After separation, these fragments can be visible by staining them with ethidium bromide followed by exposure to UV radiation.

List down the steps of DNA fingerprinting. Write down the applications of DNA fingerprinting.

DNA fingerprinting is a very quick way to analyse the differences in the DNA sequences of two individuals. It involves the identification of differences in repetitive DNA. The steps involved in DNA fingerprinting are the following: DNA isolation: DNA is extracted from the cells by using high speed centrifuge. Amplification: Many copies of the extracted DNA can be made by the use of polymerase chain reaction. Digestion of DNA: It is done by using restriction endonuclease to get DNA fragments. Separation of DNA: The DNA fragments are separated based on their charge and size using a technique called gel electrophoresis. In this technique, DNA fragments are move towards anode through a medium under an electric field due to the negative charge of these molecules. Agarose gel is used as the medium through which DNA fragments are moved. These fragments are separated according to their size due to the sieving effect of the gel. After separation, these fragments can be visible by staining them with ethidium bromide followed by exposure to UV radiation. Southern blotting: Transfer of separated DNA fragments to synthetic membrane (like nylon or nitrocellulose). Hybridisation: With the help of a radio labelled VNTR probe (small segments of DNA which help to detect the presence of a gene in a long DNA sequence) which gets attached to single stranded VNTRs having complementary nucleotide sequences. Autoradiographic exposure to X-ray films: Detection of hybridised DNA fragments by autoradiography. This shows different dark bands of different sizes. There are many applications of DNA fingerprinting. These are: It is used as a tool in forensic investigations. It is used for settling paternal dispute. It is also used for the study of evolution by determining the genetic diversities among populations.