Biotechnology Principles and Processes Test - 6

In genetic engineering, restriction enzymes (or restriction endonucleases) are used to cut DNA into smaller fragments. The cuts are always made at specific nucleotide sequences. Different restriction enzymes recognise and cut different DNA sequences. These fragments are joined together using another enzyme called ligase.

DNA ligase is a specific type of enzyme, a ligase, that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms.

The enzyme ligase is used to join vector and gene of interest to form hybrid genome that can be introduced into vectors.

A restriction enzyme (or restriction endonucleases) recognizes a specific base pair sequence in DNA called a restriction site and cleaves the DNA (hydrolyses the phosphodiester backbones) within the sequence. Restriction enzymes are widely found in prokaryotes and provide protection to the host cell by destroying foreign DNA that makes entry to it. It acts as a part of defense mechanism.

Restriction enzymes belong to a larger class of enzymes called nucleases. They are of two types: endonucleases and exonucleases.

The first restriction endonuclease discovered was Hind II. Hind II always cut DNA molecules at a particular point by recognizing a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence of Hind II.

The extraction of specific bands of DNA from agarose gels in which they are separated through electrophoresis is known as elution. There are many methods for eluting DNA from a piece of agrose.

Proteases are involved in digesting long protein chains into shorter fragments by splitting the peptide bonds that link amino acid residues.

Protein can be digested by using enzyme protease to obtain DNA. In chromosome DNA is wrapped on histone protein.

The piece of DNA obtained by gel electrophoresis contains agrose gel also. Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acids (DNA or RNA) preparations in aqueous solution. The basic procedure is that salt and ethanol are added to the aqueous solution, which forces the precipitation of nucleic acids out of solution.

A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between monomers of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules.

There are two primary classifications based on the locus of activity. Exonucleases digest nucleic acids from the ends. Endonucleases act on regions in the middle of target molecules. They are further subcategorized as deoxyribonucleases and ribonucleases. The former acts on DNA, the latter on RNA.

Restriction enzymes, such as EcoRI, are used in a wide variety of molecular genetics techniques including cloning, DNA screening and deleting sections of DNA in vitro. Restriction enzymes, like EcoRI, that generate sticky ends of DNA are often used to cut DNA prior to ligation, as the sticky ends make the ligation reaction more efficient.

Protein molecules are smaller than DNA polynucleotide. Protein and DNA can be separated by polyacrylamide gel having minute pores through which protein can pass but not the DNA.