In nature, mutations arise spontaneously but they are rare such as sickle cell anaemia. Much has been described about mutation in A Textbook of Biotechnology for Class XI. The most powerful method of introducing point mutation into a gene is known as ‘oligonucleotide (site)- directed mutagenesis.

Change in a single nucleotide base pair is called ‘point mutation’. Some useful properties in proteins (like stability in subtilisin) can be conferred by point mutation that results in substitution of selected amino acids.

Using this technique specific point of a gene can be mutated. Therefore, this method has been used to understand the function of many genes. Moreover, this technique can only be used when nucleotide sequence of gene is known.

When it has been decided which base is to be changed (e.g. A), an oligonucleotide is synthesised chemically (A). The oligonucleotide sequence has desired mutation at specific site. The oligonucleotide corresponds to the mutated nucleotide and its neighbouring regions, mainly between 15 and 20 nucleotides in length. A single stranded clone of the wild type gene is produced by using an M13 phage-based vector.

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The oligonucleotide is allowed to hybridize with the single stranded clone. The hybridization is performed under the conditions of low stringency i.e. low temperature and high salt concentration.

The oligonucleotide will base pair with wild type complementary sequence (B) and acts as primer to produce double stranded DNA by using DNA polymerase (C). The double stranded DNA consists of one wild type sequence and the other mutant sequence (C). The bacterial cells are transformed by introducing the double stranded molecule inside. The duplex DNA replicates in bacterial cell and produce either wild type or mutant plasmids.

Replication of DNA in a bacterial cell will result in a mixture of wild type and mutant double stranded DNA (D). These can be extracted and used to transform more cells. So far it is not sure whether mutation is present in desired DNA or not. To ascertain this, the DNA is isolated from bacterial cells and sequenced following the sequencing method as described earlier.

Following site-directed mutagenesis several novel proteins and enzymes have been produced and commercialized such as subtilisin, p-lactamase, tyrosyl-tRNA synthetase, etc.