Techniques in Biotechnology

Nucleic acid absorbs UV light maximally at 260 nm owing to its bases. Thus, DNA or RNA yield can be quantified based on this principle. Restriction endonucleases (type II) are one of the most important groups of enzymes for the manipulation of DNA. They are bacterial enzymes that can cut/split DNA of any source at specific sites and were first discovered in bacteria E. coli restricting the replication of bacteriophages, by cutting the viral DNA. This led to differences in the size of restriction fragments obtained due to digestion with specific restriction enzymes. A restriction map is formulated by comparing the fragment size produced in every single digestion with those from double digests. In target amplification, the nucleic acid region around the area of interest is copied many times by in vitro methods. Polymerase Chain Reaction (PCR) is the best-known and the most widely applied of the target amplification methods. Original sample DNA is digested by a restricted endonuclease, separated by agarose electrophoresis, and transferred by agarose electrophoresis, and transferred to a solid support (Nylon or Nitrocellulose membrane) followed by selective visualization of fragments using labeled hybrid.

DNA sequence has now become a routine procedure. It is the process of determination of nucleotide or base sequence of a DNA molecule/fragment. A probe is a nucleic acid whose identity is known and is used to reveal the identity or abundance of a target or sample. They are simple to prepare and are labelled with nucleotides that are radioactive, or fluorescent or have attached affinity labels. Also, by using a biotinlabelled primer, single-stranded probes can be obtained. Microarrays represent highdensity miniaturized arrays of molecular samples. The technology involves a series of probes immobilized on a glass slide as microdots, which are then hybridized with a mixture of test DNA sequences labelled with a fluorochrome.ization probes.