User:Emilyrd77/DNA shuffling

DNA shuffling
DNA shuffling, also known as molecular breeding, is an in vitro homologous recombination method to rapidly propagate mutations for directed evolution. The general approach is to fragment parent genes, allow the fragments to anneal based on sequence homology, and then amplify the recombined DNA with the polymerase chain reaction (PCR). The goal is to introduce mutations and create recombinant genes which enables the rapid increase in DNA library size.

DNA shuffling utilizes random recombination as opposed to site-directed mutagenesis in order to generate proteins with unique attributes or combinations of desirable characteristics encoded in the parent genes such as thermostability and high activity.

In 1994, Willem P.C. Stemmer published a paper on DNA shuffling to eliminate non-essential mutations in the β-lactamase gene and was able to increase the production of the antibiotic cefotaxime. Since the introduction of the technique, DNA shuffling has been applied to protein and small molecule pharmaceuticals, vaccines, gene therapy, and evolved viruses. Other techniques which yield similar results to DNA shuffling include random chimeragenesis on transient templates, random printing in vitro recombination, and staggered extension process.

History
This technique was first reported in 1994 by Willem P.C. Stemmer. He started by fragmenting the β-lactamase gene that had been amplified with PCR by using DNase I. He then completed a modified PCR reaction where primers were not employed which resulted in the annealing of homologous fragments. Finally, these fragments were amplified by PCR. Stemmer reported that the use of DNA shuffling in combination with backcrossing resulted in the elimination of non-essential mutations and an increase in the production of the antibiotic cefotaxime. He also emphasized the potential for molecular evolution with DNA shuffling. Specifically, he indicated the technique could be used to modify proteins.

DNA shuffling has since been applied to generate libraries of chimeric genes and has inspired family shuffling which is defined as the use of related genes in DNA shuffling. Additionally, this method for homologous recombination has been applied to protein and small molecule pharmaceuticals, gene therapy, vaccines, and evolved viruses.

Molecular Breeding
First, DNase I is used to fragment a set of parent genes into segments ranging from 10-50 bp to more than 1 kbp. This is followed by a PCR without primers. In the PCR, DNA fragments with sufficient overlapping homologous sequences will anneal to each other and then be extended by DNA polymerase. The PCR extension will not occur unless there are DNA sequences of high similarity. The important factors influencing the chimeric sequences synthesized in DNA shuffling are the DNA polymerase, salt concentrations, and annealing temperature. For example, the use of Taq polymerase for amplification of a 1 kbp fragment in a PCR of 20 cycles results in 33% to 98% of the products containing one or more mutations.

Multiple cycles of PCR extension can be used to amplify the fragments. The addition of primers that are designed to be complementary to the ends of the extended fragments are added to further amplify the sequences with another PCR. Primers may be chosen to have additional sequences added to their 5’ ends, such as sequences for restriction enzyme recognition sites needed for ligation into a cloning vector.

It is possible to recombine portions of the parent genes to generate hybrids or chimeric forms with unique properties, hence the term DNA shuffling.

Restriction Enzymes
Restriction enzymes are employed to fragment the parent genes. The fragments are then joined together through ligation which can be accomplished with DNA ligase. For example, if two parent genes have three restriction sites fourteen different gene hybrids can be created.

Nonhomologous Random Recombination
In order to generate segments ranging from 10-50 bp to more than 1 kb, DNase I is utilized. The ends of the fragments are made blunt by adding T4 DNA polymerase. A hairpin with a specific restriction site is then added to the mixture of fragments. Next, T4 DNA ligase is employed to ligate the fragments to form extended sequences. Finally, in order to remove the hairpin loops, a restriction enzyme is utilized.

Protein and small molecule pharmaceuticals
Since DNA shuffling enables the recombination of genes, protein activities can be enhanced. For example, DNA-shuffling has been used to increase the potency of phage-displayed recombinants on murine and human cells. The synthesis of diverse genes can also result in the production of proteins with novel attributes. The degradation of biological pollutants by proteins derived from DNA-shuffling has been accomplished. One example is the development of a recombinant E. coli strain with DNA-shuffling for the bioremediation of trichloroethylene which is less susceptible to toxic epoxide intermediates. Another example is the improvement of green fluorescent protein (GFP) where DNA-shuffling was employed to generate a 45-fold greater signal than the standard for whole cell fluorescence. Furthermore, the homologous recombination method of DNA-shuffling has been utilized to enhance the detoxification of atrazine and arsenate.

Vaccines
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. Some vaccine candidates for Plasmodium falciparum, dengue virus, encephalitic alphaviruses (including: VEEV, WEEV, and EEEV), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV) have been investigated.

Gene therapy and evolved viruses
The requirements for human gene therapies include high purity, immunity (high-titer), and stability. DNA-shuffling allows for the fabrication of retroviral vectors with these attributes. For example, this technique was applied to six ecotropic murine leukemia virus (MLV) strains which resulted in the compilation of an extensive library of recombinant retrovirus and the identification of multiple clones with increased stability. Furthermore, the application of DNA-shuffling on multiple parent adeno-associated virus (AAV) vectors was employed to generate a library of ten million chimeras. The advantageous attributes obtained include increased resistance to human intravenous immunoglobulin (IVIG) and the production of cell tropism in the novel viruses.