Editing Protein engineering (section)

=== Asexual methods ===
Asexual methods do not generate any cross links between parental genes. Single genes are used to create mutant libraries using various mutagenic techniques. These asexual methods can produce either random or focused mutagenesis.

==== Random mutagenesis ====
Random mutagenic methods produce mutations at random throughout the gene of interest. Random mutagenesis can introduce the following types of mutations: transitions, transversions, insertions, deletions, inversion, missense, and nonsense. Examples of methods for producing random mutagenesis are below.

====Error prone PCR====
Error prone PCR utilizes the fact that Taq DNA polymerase lacks 3' to 5' exonuclease activity. This results in an error rate of 0.001–0.002% per nucleotide per replication. This method begins with choosing the gene, or the area within a gene, one wishes to mutate. Next, the extent of error required is calculated based upon the type and extent of activity one wishes to generate. This extent of error determines the error prone PCR strategy to be employed. Following PCR, the genes are cloned into a plasmid and introduced to competent cell systems. These cells are then screened for desired traits. Plasmids are then isolated for colonies which show improved traits, and are then used as templates the next round of mutagenesis. Error prone PCR shows biases for certain mutations relative to others. Such as biases for transitions over transversions.<ref name=PoluriBook/>{{page needed|date=May 2017}}

Rates of error in PCR can be increased in the following ways:<ref name=PoluriBook/>{{page needed|date=May 2017}}
# Increase concentration of magnesium chloride, which stabilizes non complementary base pairing.
# Add manganese chloride to reduce base pair specificity. 
# Increased and unbalanced addition of dNTPs.
# Addition of base analogs like dITP, 8 oxo-dGTP, and dPTP.
# Increase concentration of Taq polymerase.
# Increase extension time.
# Increase cycle time.
# Use less accurate Taq polymerase.
Also see [[polymerase chain reaction]] for more information.

====Rolling circle error-prone PCR====
This PCR method is based upon rolling circle amplification, which is modeled from the method that bacteria use to amplify circular DNA. This method results in linear  DNA duplexes. These fragments contain tandem repeats of circular DNA called concatamers, which can be transformed into bacterial strains. Mutations are introduced by first cloning the target sequence into an appropriate plasmid. Next, the amplification process begins using random hexamer primers and Φ29  DNA polymerase under error prone rolling circle amplification conditions. Additional conditions to produce error prone rolling circle amplification are 1.5 pM of template DNA, 1.5 mM MnCl<sub>2</sub> and a 24 hour reaction time. MnCl<sub>2</sub> is added into the reaction mixture to promote random point mutations in the DNA strands. Mutation rates can be increased by increasing the concentration of MnCl<sub>2</sub>, or by decreasing concentration of the template DNA. Error prone rolling circle amplification is advantageous relative to error prone PCR because of its use of universal random hexamer primers, rather than specific primers. Also the reaction products of this amplification do not need to be treated with ligases or endonucleases. This reaction is isothermal.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Chemical mutagenesis====
Chemical mutagenesis involves the use of chemical agents to introduce mutations into genetic sequences. Examples of chemical mutagens follow.

Sodium bisulfate is effective at mutating G/C rich genomic sequences. This is because sodium bisulfate catalyses deamination of unmethylated cytosine to uracil.<ref name=PoluriBook/>{{page needed|date=May 2017}}

Ethyl methane sulfonate alkylates guanidine residues. This alteration causes errors during DNA replication.<ref name=PoluriBook/>{{page needed|date=May 2017}}

Nitrous acid causes transversion by de-amination of adenine and cytosine.<ref name=PoluriBook/>{{page needed|date=May 2017}}

The dual approach to random chemical mutagenesis is an iterative two step process. First it involves the ''in vivo'' chemical mutagenesis of the gene of interest via EMS. Next, the treated gene is isolated and cloning into an untreated expression vector in order to prevent mutations in the plasmid backbone.<ref name=PoluriBook/>{{page needed|date=May 2017}} This technique preserves the plasmids genetic properties.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM)====
This method has been used to create targeted  ''in vivo'' mutagenesis in yeast. This method involves the fusion of a 3-methyladenine DNA glycosylase to tetR DNA-binding domain. This has been shown to increase mutation rates by over 800 time in regions of the genome containing tetO sites.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Mutagenesis by random insertion and deletion====
This method involves alteration in length of the sequence via simultaneous deletion and insertion of chunks of bases of arbitrary length. This method has been shown to produce proteins with new functionalities via introduction of new restriction sites, specific codons, four base codons for non-natural amino acids.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Transposon based random mutagenesis====
Recently many methods for transposon based random mutagenesis have been reported. This methods include, but are not limited to the following: PERMUTE-random circular permutation, random protein truncation, random nucleotide triplet substitution, random domain/tag/multiple amino acid insertion, codon scanning mutagenesis, and multicodon scanning mutagenesis. These aforementioned techniques all require the design of mini-Mu transposons. Thermo scientific manufactures kits for the design of these transposons.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Random mutagenesis methods altering the target DNA length====
These methods involve altering gene length via insertion and deletion mutations. An example is the tandem repeat insertion (TRINS) method. This technique results in the generation of tandem repeats of random fragments of the target gene via rolling circle amplification and concurrent incorporation of these repeats into the target gene.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Mutator strains====
Mutator strains are bacterial cell lines which are deficient in one or more DNA repair mechanisms. An example of a mutator strand is the E. coli XL1-RED.<ref name=PoluriBook/>{{page needed|date=May 2017}} This subordinate strain of E. coli is deficient in the MutS, MutD, MutT DNA repair pathways. Use of mutator strains is useful at introducing many types of mutation; however, these strains show progressive sickness of culture because of the accumulation of mutations in the strains own genome.<ref name=PoluriBook/>{{page needed|date=May 2017}}

==== Focused mutagenesis ====
Focused mutagenic methods produce mutations at predetermined amino acid residues. These techniques require and understanding of the sequence-function relationship for the protein of interest. Understanding of this relationship allows for the identification of residues which are important in stability, stereoselectivity, and catalytic efficiency.<ref name=PoluriBook/>{{page needed|date=May 2017}} Examples of methods that produce focused mutagenesis are below.

====Site saturation mutagenesis====
Site saturation mutagenesis is a PCR based method used to target amino acids with significant roles in protein function. The two most common techniques for performing this are whole plasmid single PCR, and overlap extension PCR.

Whole plasmid single PCR is also referred to as site directed mutagenesis (SDM). SDM products are subjected to Dpn endonuclease digestion. This digestion results in cleavage of only the parental strand, because the parental strand contains a GmATC which is methylated at N6 of adenine. SDM does not work well for large plasmids of over ten kilobases. Also, this method is only capable of replacing two nucleotides at a time.<ref name=PoluriBook/>{{page needed|date=May 2017}}

Overlap extension PCR requires the use of two pairs of primers. One primer in each set contains a mutation. A first round of PCR using these primer sets is performed and two double stranded DNA duplexes are formed. A second round of PCR is then performed in which these duplexes are denatured and annealed with the primer sets again to produce heteroduplexes, in which each strand has a mutation. Any gaps in these newly formed heteroduplexes are filled with DNA polymerases and further amplified.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Sequence saturation mutagenesis (SeSaM)====
[[Sequence saturation mutagenesis]] results in the randomization of the target sequence at every nucleotide position. This method begins with the generation of variable length DNA fragments tailed with universal bases via the use of template transferases at the 3' termini. Next, these fragments are extended to full length using a single stranded template. The universal bases are replaced with a random standard base, causing mutations. There are several modified versions of this method such as SeSAM-Tv-II, SeSAM-Tv+, and SeSAM-III.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Single primer reactions in parallel (SPRINP)====
This site saturation mutagenesis method involves two separate PCR reaction. The first of which uses only forward primers, while the second reaction uses only reverse primers. This avoids the formation of primer dimer formation.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Mega primed and ligase free focused mutagenesis====
This site saturation mutagenic technique begins with one mutagenic oligonucleotide and one universal flanking primer. These two reactants are used for an initial PCR cycle. Products from this first PCR cycle are used as mega primers for the next PCR.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====Ω-PCR====
This site saturation mutagenic method is based on overlap extension PCR. It is used to introduce mutations at any site in a circular plasmid.<ref name=PoluriBook/>{{page needed|date=May 2017}}

====PFunkel-ominchange-OSCARR====
This method utilizes user defined site directed mutagenesis at single or multiple sites simultaneously. OSCARR is an acronym for ''one pot simple methodology for cassette randomization and recombination''. This randomization and recombination results in randomization of desired fragments of a protein. Omnichange is a sequence independent, multisite saturation mutagenesis which can saturate up to five independent codons on a gene.

====Trimer-dimer mutagenesis====
This method removes redundant codons and stop codons.

====Cassette mutagenesis====
This is a PCR based method. [[Cassette mutagenesis]] begins with the synthesis of a DNA cassette containing the gene of interest, which is flanked on either side by restriction sites. The endonuclease which cleaves these restriction sites also cleaves sites in the target plasmid. The DNA cassette and the target plasmid are both treated with endonucleases to cleave these restriction sites and create sticky ends.  Next the products from this cleavage are ligated together, resulting in the insertion  of the gene into the target plasmid. An alternative form of cassette mutagenesis called combinatorial cassette mutagenesis is used to identify the functions of individual amino acid residues in the protein of interest. Recursive ensemble mutagenesis then utilizes information from previous combinatorial cassette mutagenesis. Codon cassette mutagenesis allows you to insert or replace a single codon at a particular site in double stranded DNA.<ref name=PoluriBook/>{{page needed|date=May 2017}}