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.

What are the enzymes involved in DNA replication?

DNA replication is the method of getting two daughter strands where each strand comprises half of the DNA double helix. The process of replication requires a set of enzymes. DNA dependent DNA polymerase : It is the main enzyme that uses a DNA template to catalyse the polymerisation of deoxyribonucleotides. In prokaryotes, there are three kinds of DNA polymerases: DNA polymerase I, II and III. In eukaryotes, five categories of these enzymes are identified that are alpha, beta, gamma, sigma and epsilon. Helicase: It unwinds the DNA strand, i.e., separates the two DNA strands from one point that leads to the formation of replication fork. Topoisomerase: The unwinding of DNA creates a tension in the DNA strands that gets released by the enzyme topoisomerase. DNA ligase: It facilitates the joining of DNA strand together by catalysing the formation of phosphodiester bond. It plays a role in repairing single strand breaks in duplex DNA.

Why are both the strands of DNA not copied during transcription?

During transcription only one strand of DNA double helix acts as template and copied. If both the DNA strands act as templates, they would code for RNA molecule with different sequences, and in turn, if they code for proteins, the sequence of amino acids in the proteins would be different. Hence, one segment of the DNA would be coding for two different proteins, and this would complicate the genetic information transfer machinery. The two antiparallel strands of DNA have a complementary sequence of bases. During transcription when the RNA strand is formed, it will have a complementary sequence to the DNA template. When two RNA strands are formed from the two DNA template strands, these two RNA strands will be complementary to each other and will form double-stranded RNA, which cannot undergo translation.