Mutagenesis is defined as the change in the genetic information of an organism in a stable manner by the use of physical and chemical mutagens. It was developed by Charlotte Auerbach. She was the first women scientist to study on the effect of chemical mutagens.

This immediately found use as a genetic tool to induce mutations in specific ways which in turn can be used to determine the phenotype of the organism, the function of the genes and even the nucleotides.

There are different types of Mutagenesis like

1. Directed Mutagenesis

2. Random Mutagenesis

3. Insertional Mutagenesis
• Signature Tagged Mutageneis
• Virus insertional Mutagenesis

4. PCR Mutagenesis
• Site Directed Mutagenesis
• Mismatched Mutagenesis
• 5'Add On Mutagenesis
• Cassette Mutagenesis


1. Directed Mutagenesis

Directed Mutagenesis is defined as the change in amino acid coding at the DNA level. A characterized 3D structure of the protein using X Ray crystallography and other analytical procedures helps in determining which amino acids of protein should be changed to attain specific property. However,this may not be possible for most of the proteins and hence a trial and error strategy is used to make changes in the nucleotides to yield a particular change in the protein. The encoded protein is then tested for the desired change in the protein.

Different types of Directed Mutagenesis are:

a. Oligonucleotide Directed Mutagenesis With M13 DNA

b. Oligonucleotide directed Mutagenesis with Plasmid DNA.

c. PCR Amplified oligonucleotide Directed Mutagenesis

Advantages :
- Mutation rates is high
- All mutations can be induced
- Systematic and detailed investigation of the targeted mutation can be made.


2. Random Mutagenesis

Random Mutagenesis is also known as Directed Evolution or Molecular breeding. In this the gene is non specifically changed at one or more codon levels to produce a mixture of mutated genes which in turn can be selected and screened for the genes with desired catalytic activity. The oligonucleotide primer is a heterogeneous set of DNA sequences to produce a series of mutation in the defined portion of the target gene.

Advantages: The role of an amino acid in the working of the protein is not required.
Since a range of mutants are produced in this process some interesting and useful proteins may be generated

3. Insertional Mutagenesis:

Insertional Mutagenesis is produced by the insertion of one or more bases and can be produced
1. Natural method
2. Mediated by Virus or transposon(Signature tagged Mutagenesis) or
3. Artificially in the lab for research purpose

a. Signature Tagged Mutagenesis : Is used to study the function of genes using transposons.A transposon such as Drosophila melanogaster P -Element is made to integrate randomly in the genome of an organism. The mutants are then screened for any unusual phenotype.Any such phenotype is found suggests that the transposon has caused the inactivation of the phenotype related to that gene. Since the sequence of transposon is known the whole genome can be sequenced to identify the gene or PCR can be used to amplify the specific gene.

b. Virus Insertional Mutagenesis: Is caused by replication competent virus.The virus inserts viral oncogene near Cellular myc gene which are generally turned off in a cell and when turned on pushes the cell into G1 of the cell cycle which allows even the viral gene to get replicated.Latent tumors are produced after many replication at the site of viral gene.Eg. Avian Leukosis virus causes disease using insertional mutagenesis.

Advantages:
- Can produce easily tractable Mutations.
- Can produce large number of mutants at low cost and high speed

4. PCR Mutagenesis :

Is used to change the nucleotide sequence of the DNA.The method can be used to alter amino acids to test the function of domains in a protein and to asses the function of a promoter.

Different ways of introducing mutations in PCR are :
a. Site Directed Mutagenesis: As the name suggests it introduces mutation at a specific location on the DNA strand.The primers used are altered at one or more nucleotides.

b. Mismatched Mutagenesis :Is used with focus on a single amono acid and is useful in checking missense mutation in a known gene of disease or in determining the function of a particular amino acid in the protein.

c. 5'Add On Mutagenesis is produced by adding a new sequence or chemical group to the 5'end of the PCR product.The primers are produced in a specific manner

- 3' end of the primer matches the sequence of PCR product.
- 5' end contains the novel sequence
- Suitable restriction site
- Addition of a functional sequence (promoter sequence)
- Modified nucleotide that contains labeled group, biotinlyated, or fluorophore


d. Cassette Mutagenesis: Is used to introduce multiple mutations into the DNA sequence.Using blunt ended DNA at the site of mutation a 3 base pair direct terminal repeat is created.The mutagenic codon cassette has a head to head Sapl site to remove all the DNA except the mutated one.

Advantages:
PCR based methods are useful in the study of specific mutations in the DNA which in turn is useful in the study of different aspects of the protein function.

Limitations
- Mutated DNA is hard to replicate as the competent cell to be used are expensive.
- Screening is tedious.
- Requires sequencing to confirm mutation.
- Specific primers are required.

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