DNA shuffling

DNA shuffling, also known as molecular breeding, is an in vitro random recombination method to generate mutant genes for directed evolution and to enable a rapid increase in DNA library size. Three procedures for accomplishing DNA shuffling are molecular breeding which relies on homologous recombination or the similarity of the DNA sequences, restriction enzymes which rely on common restriction sites, and nonhomologous random recombination which requires the use of hairpins. As a result of the random recombination, DNA shuffling is able to produce proteins with new qualities or multiple advantageous features derived from the parent genes.

DNA shuffling by molecular breeding was first reported in 1994 by Willem P. C. Stemmer. He then completed a modified PCR reaction where primers were not employed which resulted in the annealing of homologous fragments or fragments with similar sequences. Specifically, he indicated the technique could be used to modify proteins. Additionally, DNA shuffling has been applied to protein and small molecule pharmaceuticals, bioremediation, gene therapy, vaccines, and evolved viruses.

First, DNase I is used to fragment a set of parent genes into segments of double stranded DNA ranging from 10 50 bp to more than 1 kbp. The PCR extension will not occur unless there are DNA sequences of high similarity. The disadvantage of molecular breeding is the requirement for the similarity between the sequences, which has inspired the development of other procedures for DNA shuffling. The main disadvantage is the requirement of common restriction enzyme sites. The ends of the fragments are made blunt by adding T4 DNA polymerase. Hairpins with a specific restriction site are then added to the mixture of fragments.

DNA shuffling has also been used to improve the degradation of biological pollutants.

The ability to select desirable recombinants with DNA shuffling has been used in combination with screening strategies to enhance vaccine candidates against infections with an emphasis on improving immunogenicity, vaccine production, stability, and cross reactivity to multiple strains of pathogens. DNA shuffling allows for the fabrication of retroviral vectors with these attributes. The advantageous attributes obtained include increased resistance to human intravenous immunoglobulin (IVIG) and the production of cell tropism in the novel viruses. These random primers are annealed to template DNA and are then extended by the Klenow fragment. The main advantages of StEP are the simplicity of the method and the lack of fragment purification. The disadvantages of StEP include that it is time consuming and requires sequence homology.