DNA shuffling, also known as molecular breeding, is an

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...

random recombination method to generate mutant

genes In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...

for

directed evolution Directed evolution (DE) is a method used in protein engineering that mimics the process of natural selection to steer proteins or nucleic acids toward a user-defined goal. It consists of subjecting a gene to iterative rounds of mutagenesis (cre ...

and to enable a rapid increase in DNA library size. Three procedures for accomplishing DNA shuffling are molecular breeding which relies on

homologous recombination Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may ...

or the similarity of the DNA sequences,

restriction enzymes A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...

which rely on common

restriction sites Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences (beca ...

, and nonhomologous random recombination which requires the use of hairpins. In all of these techniques, the parent genes are fragmented and then recombined. DNA shuffling utilizes random recombination as opposed to

site-directed mutagenesis Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesi ...

in order to generate proteins with unique attributes or combinations of desirable characteristics encoded in the parent genes such as

thermostability In materials science and molecular biology, thermostability is the ability of a substance to resist irreversible change in its chemical or physical structure, often by resisting decomposition or polymerization, at a high relative temperature. ...

and high activity. The potential for DNA shuffling to produce novel proteins is exemplified by the figure shown on the right which demonstrates the difference between point mutations, insertions and deletions, and DNA shuffling. Specifically, this figure shows the use of DNA shuffling on two parent genes which enables the generation of recombinant proteins that have a random combination of sequences from each parent gene. This is distinct from

point mutations A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequence ...

in which one nucleotide has been changed, inserted, or deleted and insertions or deletions where a sequence of nucleotides has been added or removed, respectively. 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. In 1994, Willem P.C. Stemmer published the first paper on DNA shuffling. Since the introduction of the technique, DNA shuffling has been applied to protein and small molecule pharmaceuticals,

bioremediation Bioremediation broadly refers to any process wherein a biological system (typically bacteria, microalgae, fungi, and plants), living or dead, is employed for removing environmental pollutants from air, water, soil, flue gasses, industrial effluent ...

vaccines A vaccine is a biological preparation that provides active acquired immunity The adaptive immune system, also known as the acquired immune system, is a subsystem of the immune system that is composed of specialized, systemic cells and pro ...

gene therapy Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DN ...

, and evolved viruses. Other techniques which yield similar results to DNA shuffling include

random chimeragenesis on transient templates Random Chimera (genetics), chimeragenesis on transient templates (RACHITT) is a method to perform molecular mutagenesis at a high recombination rate. For example, RACHITT can be used to generate increased rate and extent of biodesulfurization of die ...

(RACHITT), random printing in vitro recombination (RPR), and the

staggered extension process The staggered extension process (also referred to as StEP) is a common technique used in biotechnology and molecular biology to create new, mutated genes with qualities of one or more initial genes. The technique itself is a modified polymerase cha ...

(StEP).

History

DNA shuffling by molecular breeding was first reported in 1994 by Willem P.C. Stemmer. He started by fragmenting the β-lactamase gene that had been amplified with the

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...

(PCR) by using

DNase I Deoxyribonuclease I (usually called DNase I), is an endonuclease of the DNase family coded by the human gene DNASE1. DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding ...

, which randomly cleaves DNA. 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. Finally, these fragments were amplified by PCR. Stemmer reported that the use of DNA shuffling in combination with

backcrossing Backcrossing is a crossing of a hybrid with one of its parents or an individual genetically similar to its parent, to achieve offspring with a genetic identity closer to that of the parent. It is used in horticulture, animal breeding, and product ...

resulted in the elimination of non-essential

mutations In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...

and an increase in the production of the

antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of ...

cefotaxime Cefotaxime is an antibiotic used to treat a number of bacterial infections in human, other animals and plant tissue culture. Specifically in humans it is used to treat joint infections, pelvic inflammatory disease, meningitis, pneumonia, urin ...

. He also emphasized the potential for

molecular evolution Molecular evolution is the process of change in the sequence composition of cellular molecules such as DNA, RNA, and proteins across generations. The field of molecular evolution uses principles of evolutionary biology and population genetics ...

with DNA shuffling. Specifically, he indicated the technique could be used to modify proteins. DNA shuffling has since been applied to generate libraries of hybrid or

chimeric gene Chimeric genes (literally, made of parts from different sources) form through the combination of portions of two or more coding sequences to produce new genes. These mutations are distinct from fusion genes which merge whole gene sequences into ...

s and has inspired family shuffling which is defined as the use of related genes in DNA shuffling. Additionally, DNA shuffling has been applied to protein and small molecule pharmaceuticals, bioremediation, gene therapy, vaccines, and evolved viruses.

Procedures

Molecular breeding

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. This is followed by a PCR without primers. In the PCR, DNA fragments with sufficiently overlapping sequences will anneal to each other and then be extended by

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...

. The PCR extension will not occur unless there are DNA sequences of high similarity. The important factors influencing the sequences synthesized in DNA shuffling are the DNA polymerase, salt concentrations, and annealing temperature. For example, the use of

Taq polymerase ''Taq'' polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism ''Thermus aquaticus,'' from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to ''Taq'' or ''Ta ...

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 on to their 5’ ends, such as sequences for

restriction enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...

recognition sites which are needed for ligation into a

cloning vector A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or ...

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

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 DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organ ...

. For example, if two parent genes have three restriction sites fourteen different full-length gene hybrids can be created. The number of unique full-length hybrids is determined by the fact that a gene with three restriction sites can be broken up into four fragments. Thus, there are two options for each of the four positions minus the combinations that would recreate the two parent genes yielding 2⁴ - 2 = 14 different full-length hybrid genes. The main difference between DNA shuffling with restriction enzymes and molecular breeding is molecular breeding relies on the homology of the sequences for the annealing of the strands and PCR for extension whereas by using restriction enzymes, fragment ends that can be ligated are created. The main advantages of using restriction enzymes include control over the number of recombination events and lack of PCR amplification requirement. The main disadvantage is the requirement of common restriction enzyme sites.

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. Blunting the fragments is important for combining the fragments as incompatible sticky-ends, or overhangs, prevent end joining. Hairpins with a specific restriction site are then added to the mixture of fragments. Next, T4 DNA ligase is employed to ligate the fragments to form extended sequences. The ligation of the hairpins to the fragments limits the length of the extended sequences by preventing the addition of more fragments. Finally, in order to remove the hairpin loops, a restriction enzyme is utilized. Nonhomologous random recombination differs from molecular breeding as homology of the ligated sequences is not necessary which is an advantage. However, because this process recombines the fragments randomly it is probable that a large fraction of the recombined DNA sequences will not have the desired characteristics which is a disadvantage. Nonhomologous random recombination also differs from the use of restriction enzymes for DNA shuffling as common restriction enzyme sites on the parent genes are not required and the use of hairpins is necessary which demonstrates an advantage and disadvantage of nonhomologous random recombination over the use of restriction enzymes, respectively.

Applications

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 recombinant

interferons Interferons (IFNs, ) are a group of signaling proteins made and released by host cells in response to the presence of several viruses. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten the ...

on murine and human cells. Additionally, the improvement of

green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...

(GFP) was accomplished with DNA shuffling by molecular breeding as a 45-fold greater signal than the standard for whole cell fluorescence was obtained. Furthermore, the synthesis of diverse genes can also result in the production of proteins with novel attributes. Therefore, DNA shuffling has been used to develop proteins to detoxify chemicals. For example, the homologous recombination method of DNA shuffling by molecular breeding has been utilized to enhance the detoxification of

atrazine Atrazine is a chlorinated herbicide of the triazine class. It is used to prevent pre-emergence broadleaf weeds in crops such as maize (corn), soybean and sugarcane and on turf, such as golf courses and residential lawns. Atrazine's primary manufa ...

and

arsenate The arsenate ion is . An arsenate (compound) is any compound that contains this ion. Arsenates are salts or esters of arsenic acid. The arsenic atom in arsenate has a valency of 5 and is also known as pentavalent arsenic or As(V). Arsenate resem ...

Bioremediation

DNA shuffling has also been used to improve the degradation of biological pollutants. Specifically, a recombinant E. coli strain has been created with the use of DNA shuffling by molecular breeding for the bioremediation of

trichloroethylene The chemical compound trichloroethylene is a halocarbon commonly used as an industrial solvent. It is a clear, colourless non-flammable liquid with a chloroform-like sweet smell. It should not be confused with the similar 1,1,1-trichloroethane, ...

(TCE), a potential

carcinogen A carcinogen is any substance, radionuclide, or radiation that promotes carcinogenesis (the formation of cancer). This may be due to the ability to damage the genome or to the disruption of cellular metabolic processes. Several radioactive substan ...

, which is less susceptible to toxic epoxide intermediates.

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 Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. It may be wanted or unwanted: * Wanted immunogenicity typically relates to vaccines, where the injectio ...

, vaccine production, stability, and

cross-reactivity Cross-reactivity, in a general sense, is the reactivity of an observed agent which initiates reactions outside the main reaction expected. This has implications for any kind of test or assay, including diagnostic tests in medicine, and can be a ...

to multiple strains of pathogens. Some vaccine candidates for

Plasmodium falciparum ''Plasmodium falciparum'' is a Unicellular organism, unicellular protozoan parasite of humans, and the deadliest species of ''Plasmodium'' that causes malaria in humans. The parasite is transmitted through the bite of a female ''Anopheles'' mosqu ...

dengue virus ''Dengue virus'' (DENV) is the cause of dengue fever. It is a mosquito-borne, single positive-stranded RNA virus of the family ''Flaviviridae''; genus ''Flavivirus''. Four serotypes of the virus have been found, a reported fifth has yet to be co ...

, encephalitic alphaviruses (including: VEEV,

WEEV Andrew Alan Escher Auernheimer ( ; born ), best known by his pseudonym weev, is an American computer hacker and professional Internet troll. Affiliated with the alt-right, the Southern Poverty Law Center has described him as being a neo-Nazi, w ...

, and EEEV), human immunodeficiency virus-1 (HIV-1), and

hepatitis B virus ''Hepatitis B virus'' (HBV) is a partially double-stranded DNA virus, a species of the genus ''Orthohepadnavirus'' and a member of the ''Hepadnaviridae'' family of viruses. This virus causes the disease hepatitis B. Disease Despite there bein ...

(HBV) have been investigated.

Gene therapy and evolved viruses

The requirements for human gene therapies include high purity, high-

titer Titer (American English) or titre (British English) is a way of expressing concentration. Titer testing employs serial dilution to obtain approximate quantitative information from an analytical procedure that inherently only evaluates as positiv ...

, and stability. DNA shuffling allows for the fabrication of retroviral vectors with these attributes. For example, DNA shuffling with molecular breeding was applied to six

ecotropic Ecotropism or ecotropic (from ''eco'' – hearth and ''tropic'' – to turn towards) refers to the philosophy that for human culture to be healthy, it must exist as in an ecological niche and thereby relate appropriately with all the fields of force ...

murine leukemia virus The murine leukemia viruses (MLVs or MuLVs) are retroviruses named for their ability to cause cancer in murine (mouse) hosts. Some MLVs may infect other vertebrates. MLVs include both exogenous and endogenous viruses. Replicating MLVs have a pos ...

(MLV) strains which resulted in the compilation of an extensive library of recombinant

retrovirus A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase ...

and the identification of multiple clones with increased stability. Furthermore, the application of DNA shuffling by molecular breeding on multiple parent

adeno-associated virus Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus ''Dependoparvovirus'', which in turn belongs to the family ''Parvoviridae''. They are small (approximately 26 nm in di ...

(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 A tropism is a biological phenomenon, indicating growth or turning movement of a biological organism, usually a plant, in response to an environmental stimulus. In tropisms, this response is dependent on the direction of the stimulus (as oppose ...

in the novel viruses.

Comparison to other techniques

While DNA shuffling has become a useful technique for random recombination, other methods including RACHITT, RPR, and StEP have also been developed for this purpose. Below are some advantages and disadvantages of these other methods for recombination.

RACHITT

In RACHITT, fragments of single stranded (ss) parent genes are annealed onto a ss template resulting in decreased mismatching which is an advantage. Additionally, RACHIIT enables genes with low sequence similarity to be recombined. However, a major disadvantage is the preparation of the ss fragments of the parent genes and ss template.

RPR

RPR makes use of random primers. These random primers are annealed to template DNA and are then extended by the

Klenow fragment The Klenow fragment is a large protein fragment produced when DNA polymerase I from '' E. coli'' is enzymatically cleaved by the protease subtilisin. First reported in 1970, it retains the 5' → 3' polymerase activity and the 3’ → 5’ ex ...

. Next, the templates are removed and the fragments are assembled by homology in a process similar to PCR. Some major benefits include the smaller requirement for parent genes due to the use of ss templates and increased sequence diversity by mispriming and misincorporation. One disadvantage of RPR is the preparation of the template.

StEP

In StEP, brief cycles of primer annealing to a template and extension by polymerase are employed to generate full-length sequences.{{cite book , vauthors = Aguinaldo AM, Arnold FH , title = Directed Evolution Library Creation , chapter = Staggered extension process (StEP) in vitro recombination , volume = 231 , pages = 105–110 , date = 2003 , pmid = 12824608 , doi = 10.1385/1-59259-395-X:105 , publisher = Humana Press , isbn = 978-1-59259-395-8 , series = Methods in Molecular Biology , veditors = Arnold FH, Georgiou G , place = Totowa, NJ 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.

References

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