Question

. You want to improve your protein to have desired properties by introducing random mutations only into the selected spot (hatched in the below diagram), but not whole regions, of the protein. What kind of mutation technique will you use to generate protein library to introduce random mutations to only the selected regions? Please explain your chosen mutation method in detail, including your primer design and PCR strategy

Answer 1

Creation of library of proteins needs to target a controlled level of randomization to specific positions within the DNA sequence. based on the incorporation of partially randomized synthetic DNA cassettes ,these methods involve the synthesis of a mixtures of DNA molecules , they are introduced into genes with the help of Polymerase Chain Reaction ( PCR) or direct cloning. These method is based on the introduction of modified genes at specific positions within the synthetic DNA.

Mostly the modified gene mutation leading to a change in its product is not possible in humans yet. Nevertheless, The general method can be described as follows:

A mutated DNA molecule gets inserted into the chromosome of the cell randomly and yielding rare occurrence of homologous recombination and the required effects. Although these gene targeting event is rare , this method is used for any gene to get altered or inactivated.

in the first step, a DNA fragment containing a desired mutant gene is inserted into a vector. They are then introduced into a special line, which contains embryo-derived stem cells, which are called embryonic stem cells or ES cells. The ES cells are grown in cell culture and can produce different tissue types. After a waiting period of cell proliferation, the rare colonies of cells in which a homologous recombination event is likely to have caused a gene replacement to occur are isolated. With the process of PCR or southern blotting the correct colonies among these are identified .These contain recombinant DNA sequences in which the desired effect that is inserted fragment has replaced all or part of one copy of the normal gene. In the second step, with the help of a micropipette individual cells from the identified colony are taken up and injected into an early embryo. Thus a set of transfected embryo-derived stem cells are found which are then collaborate with the cells of the host embryo leading to the production of the desired protein.

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