Random point mutations

In DNA shuffling starting from a single gene as the parent template, diversity originates from random point mutations, due to the limited fidelity of polymerases... 

A library of parent DNA sequences encoding for the desired protein is chosen. Sequence diversity is created or increased through a mutagenesis step, either by introduction of random point mutations through error-prone PCR or by recombination of DNA fragments such as DNA shuffling or RACHITT. 

Of all the methods for introduction of random mutations into genes, pathways, and organisms, only those have been discussed in detail here that are frequently used in conjunction with directed evolution protocols. Several approaches exist which represent in essence a hybrid between random point mutation and recombination these approaches are covered in Section 11.4 and listed in Table 11.2. 

The germ-line theory According to this model the primary structure of every V region an animal can produce is encoded in its DNA. Every vertebrate genome contains many thousands of different V genes with divergence due to natural selection of favourable random point mutations, duplications and recombinations. 

Figure 2 Genetic operators used to create a population of children chromosomes from a population of parent chromosomes, (a) Single-point mutation. A gene to he mutated is selected at random, and its value is modified, (b) One-point crossover. The crossover point is selected randomly, and the genes are exchanged between the two parents. Two children are created, each having genes from both parents.

Fig. 4.1. Schematic representation of die StEP process using two parental DNA sequences. (1) Denatured template DNAs are primed widi defined primers. (2) The partially extended primers produced by very brief annealing/extension randomly reanneal to different parent sequences (template switching). (3) Novel recombinants are created through multiple cycles of annealing/extension and strand switelling, hi principle, StEP is also an error-prone amplification process dial introduces additional point mutations (white circles).

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