What is a random change in the sequence of DNA?

DTATEGF Construction

DNA shuffling and DNA cloning techniques were used to synthesize and assemble the hybrid genes encoding the single-chain DTATEGF (Figure 15.2) (Vallera et al., 2002; Oh et al., 2009). From the 5′ to 3′ end, the assembled fusion gene contained an Nco1 restriction site, an ATG initiation codon, the first 389 amino acids of the DT molecule (DT390), the seven-amino-acid EASGGPE linker, the genes for human EGF and 135-ATF from uPA linked by 20-amino-acid segment of human muscle aldolase, and a XhoI restriction site (Huang et al., 2012). The resulting NcoI/XhoI fragment gene was spliced into the pET21d expression vector under the control of an isopropyl-h-D-thiogalactopyranoside-inducible T7 promoter. Verification that the gene was correct in sequence and had been cloned in frame was confirmed using DNA sequencing analysis. The monospecific targeted toxins DTAT and DTEGF were created using the same techniques (Vallera et al., 2002; Oh et al., 2009). A Novagen pET expression system was used for protein expression and purification from inclusion bodies. Fast protein liquid chromatography-ion exchange chromatography (Q Sepharose Fast Flow, Sigma) was used to purify refolded proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining was performed to determine protein purity.

2.2.1 DNA shuffling and family shuffling

Stemmer introduced DNA shuffling, the first homologous recombination method, in 1994 [5, 6]. DNA shuffling involves the digestion of a gene by DNaseI into random fragments, and the reassembly of those fragments into a full-length gene by primerless PCR: the fragments prime on each other based on sequence homology, and recombination occurs when fragments from one copy of a gene anneal to fragments from another copy, causing a template switch, or crossover event. This method was used to fragment and recreate a single gene, to recombine a group of point mutants, and to recombine several related genes. The reassembly process introduces point mutations at a rate similar to error-prone PCR, due to misincorporations by the DNA polymerase. These mutations add to the diversity of the mutant library, and any unnecessary mutations can later be eliminated by backcrossing to parent or wild-type sequences. If necessary, use of a high fidelity DNA polymerase allows the rate of random point mutations to be reduced drastically [60]. Several years after the introduction of DNA shuffling, the method was applied to the recombination of a family of related genes from various species. This new method, termed family shuffling, applied DNA shuffling to a group of naturally occurring homologous genes rather than laboratory-created mutants. Crameri et al found that family shuffling significantly accelerated the rate of functional enzyme improvement in a single recombination-selection cycle [61]. Although they are powerful methods, DNA shuffling and family shuffling are not without limitations. Shuffling methods require the presence of zones of relatively high sequence homology surrounding regions of diversity [6]. Additionally, significant biases are found in where crossover events occur and in which parents are involved: crossover tends to occur in regions of higher homology, and among parents which share greater sequence identity [62]. Bias is also introduced by nonrandom gene fragmentation by the DNaseI enzyme [63]. All of these factors limit the diversity created in a shuffled library. In extreme cases, lack of homology among parents can lead to the majority of reconstructed “shuffled” sequences entirely representing a single parent [64].

Numerous homologous gene recombination methods have been designed to address the limitations of family shuffling. Kikuchi et al described a method for gene shuffling using endonuclease digestion at restriction sites, rather than DNaseI digestion; however, sequence homology surrounding the digested restriction sites is still required for overlap extension to occur [64, 65]. Degenerate oligonucleotide gene shuffling (DOGS) utilizes a PCR reaction with degenerate-end, complementary primer pairs to shuffle genes with limited sequence similarity and G + C content [64]. Additionally, by modifying primer extension conditions the progeny can be biased towards one or more of the parent genes [64].

2.1.5 In vitro DNA shuffling of GPCR sequences to generate highly diverse chimeric receptor libraries

In vitro DNA shuffling is used to generate chimeras from two receptor sequences. We have recently adapted and optimized the staggered extension process (StEP; Fig. 4.2A) for generation of chimeric libraries starting from the rNTR1-D03 and a mutagenized artificial receptor sequence, rNTR1-M30 or rNTR1-M303 (Schlinkmann, Hillenbrand, et al., 2012). StEP is a PCR-based approach (Aguinaldo & Arnold, 2002; Zhao & Zha, 2006), in which two or more different sequences are used as input templates. By using a very short combined annealing and extension step at a suboptimal DNA-polymerase elongation temperature, the amplification primers are only extended by a few nucleotides per StEP-PCR cycle. After the subsequent denaturation step, the primers are further extended, until eventually a full-length receptor sequence is obtained. Most importantly, by using two or more different input templates, the growing primer fragment can switch templates between two PCR cycles, and thus accumulates mutations from two different templates in one chimeric receptor sequence.

For shuffling of two GPCR variants of approximately 1200 bp, 10 ng of each plasmid template DNA is mixed per 50 μl PCR using 2 units VentR® DNA Polymerase (NEB) and 30 pmol each of the two flanking primers, introducing a restriction site. The choice of DNA polymerase greatly affects shuffling efficiency, and the following aspects should be considered: High fidelity is required to avoid undesired mutations as a result of high number of StEP-PCR cycles. To yield short recombination distances, a slower DNA polymerase is preferred, for example VentR® (NEB, ∼ 1000 bp/min) over Phusion® DNA polymerase (Finnzymes, 1000 bp/15 s).

The amplification yield in a StEP reaction is comparably low, and further PCR amplification of a single StEP reaction should be avoided, as it does not increase diversity. With a theoretical diversity of about 107, 12 reactions are setup in parallel to increase the product amount while simultaneously generating high diversity. StEP shuffling is performed for 125 PCR cycles on a Biometra T3 thermocycler (heating rate of 2 °C/s) with 30 s denaturation at 94 °C and 6 s annealing/elongation at 50 °C per cycle and 2 min of initial denaturation. A final and extended elongation step should be omitted, as it could lead to amplification of the template sequences without shuffling.

StEP is a delicate PCR-based process, reacting strongly to small changes in reaction conditions. The most important parameters for optimization and troubleshooting are duration and temperature of annealing and elongation, which are key determinants of shuffling efficiency. Under these conditions, recombination events within 30-bp distance are obtained. Importantly, the differences in heating and cooling rates between thermocyclers strongly influence the recombination efficiency and yield of the StEP process, and should be controlled and adjusted together with the elongation conditions. With slow heating and cooling rates (2 °C/s), the actual window of DNA-polymerase activity is longer than defined by the annealing and elongation cycle (here 6 s per cycle), compared to thermocyclers with fast heating rates (up to 6 °C/s). Under these conditions, elongation times might have to be extended to allow sufficient product formation.

VentR® and Phusion DNA polymerases both exhibit 3′- to 5′-proofreading exonuclease activity, which, for the case that an incorporated mutation locates at the 3′-end of the growing fragment, could lead to correction by the polymerase proofreading activity after template switching. Thus, a DNA polymerase lacking 3′- to 5′-proofreading exonuclease activity, for example Deep VentR™ (exo–) (NEB), might at first seem attractive with respect to recombination efficiency, but it resulted in high amounts of PCR side products in our experiments and was thus not used.

Furthermore, despite the presence of 2 mM MgSO4 in the PCR buffer, we observed that a further increase to a final of 4 mM MgSO4 positively affected the reaction yield, probably by stabilizing DNA-polymerase complexes after mismatches.

The shuffled StEP product is digested with DpnI to minimize the carryover of input templates. The StEP product should be purified from a preparative agarose gel, as PCR side products are common to StEP reactions. The purified product is digested with the corresponding restriction enzymes and purified. At least 3 μg of final StEP product should be obtained for subcloning of a product with a theoretical diversity of 107.

Shuffling by StEP is an easy and fast technique to generate a chimeric library from two or more target sequences. However, mutations close in sequence (3–30 bp) are inefficiently separated by StEP, and sequences with coupled mutations are overrepresented, compared to the recombined sequences. If more than two templates are shuffled, the apparent recombination efficiency can suffer from a “dilution effect” (Fig. 4.2B): If 10 individual point mutants of a given receptor are used as input templates for StEP shuffling, one sequence will contain a particular mutation, while nine templates contain the wild-type codon in the respective position. Statistically, eight of nine recombination events will shuffle wild type against wild type and the accumulation of mutations in one shuffled sequence is consequently low.

Alternatively, an artificial receptor sequence combining all mutations of interest can be synthesized and shuffled against the wild-type sequence for an mutant to wild-type ratio of 1:1 (e.g., Schlinkmann, Hillenbrand, et al., 2012). Evidently, the above effect can be easily exploited to direct and influence recombination by adjustment of template ratios and template design.

The selection output from a diverse StEP-library can be readily subjected to a further StEP shuffling by plasmid DNA isolation from the selected cell pool.

DNA Shuffling

Natural selection works on new sequences generated both by mutation and recombination. DNA shuffling is a method of artificial evolution that includes the creation of novel mutations as well as recombination. The gene to be improved is cut into random segments around 100 to 300 base pairs long. The segments are then reassembled by using a suitable DNA polymerase with overlapping segments or by using some version of overlap PCR (see Chapter 4). This recombines segments from different copies of the same gene (Fig. 11.9).

Mutations may be introduced in several ways, including the standard mutagenesis procedures already described. In addition, the DNA segments may be generated using error-prone PCR (see earlier discussion) instead of by using restriction enzymes. Alternatively, mutations may be introduced during the reassembly procedure itself by using a DNA polymerase that has impaired proofreading capability. The result is a large number of copies of the gene, each with several mutations scattered at random throughout its sequence. The final shuffled and mutated gene copies must then be expressed and screened for altered properties of the encoded protein.

A more powerful variant of DNA shuffling is to start with several closely related (i.e., homologous) versions of the same gene from different organisms. The genes are cut at random with appropriate restriction enzymes and the segments mixed before reassembly. The result is a mixture of genes that have recombined different segments from different original genes (Fig. 11.10). Note that the reassembled segments keep their original natural order. For example, several related β-lactamases from different enteric bacteria have been shuffled. The shuffled genes were cloned onto a plasmid vector and transformed into host bacteria. The bacteria were then screened for resistance to selected β-lactam antibiotics. This approach yielded improved β-lactamases that degraded certain penicillins and cephalosporins more rapidly and so made their host cells up to 500-fold more resistant to these β-lactam antibiotics.

In DNA shuffling, the coding sequence for a protein is rearranged in the hope of generating novel or improved activities. Mutations may also be introduced during the procedure to provide more variation.

Vector Optimization Using DNA Shuffling and Directed Evolution

The rapidly developing fields of DNA shuffling and directed evolution provide an excellent means to select mutant AAV vectors that exhibit a range of specific properties. For example, Schaffer's laboratory recently introduced random changes in a single AAV2 capsid protein using error prone PCR and then selected those mutants that exhibited specific properties. By combining error prone PCR with directed evolution, they were able to identify unique AAV2 variants that exhibited marked reduction in heparin binding or evaded AAV2 immunity in vivo. These studies provided the proof-of-principle that directed evolution could be successfully applied to AAV.

Another means to introduce substantial diversity into AAV capsid proteins utilizes the rapidily advancing technique of DNA shuffling. Through this method, enormous diversity can be generated across a wide range of different AAV capsid proteins. Essentially, libraries of hybrid genes can be generated by random fragmentation of a pool of related genes, followed by reassembly of the fragments in a self-priming polymerase reaction. When this technique is combined with directed evolution, both single and multigene traits can be evolved rapidly even though they might require many mutations to achieve the improved phenotypes. As mentioned previously, we have no a priori knowledge of those properties that would optimize transduction of chronic seizure-exposed tissue. Thus, any attempt at rational mutagenesis would proceed in the absence of a guiding rationale. In contrast, DNA shuffling combined with directed evolution allows selection of those mutants that actually fulfill the desired phenotypic properties. We currently are pursuing this approach in order to identify the optimal AAV vector for anti-seizure gene therapy.

9.20.5 Conclusions

Since its slow beginnings, and its renaissance after the introduction of DNA shuffling in 1994, the technique of directed molecular evolution has steadily increased in both popularity and number of applications. Many inventive molecular techniques and the incorporation of directed, or rational, approaches in order to focus on the search of sequence space have led to several major technical advances. Directed molecular evolution is now widely used in many industries, not least of which is the pharmaceutical industry, in which the use of evolved enzymes to generate enantiopure starting materials or to contribute to chemoenzymatic syntheses is becoming increasingly popular. The technical approaches and examples of the application should serve to provide the reader with an appreciation of both the potential and the limitations of utilizing this technique to improve the already significant catalytic benefit that enzymes can provide in a wide range of applications.

DNA SHUFFLING

A newly introduced method for generating mutations in vitro is known as DNA shuffling (Fig. 6). This method has been applied to interleukin 1β (IL-1β) (Stemmer, 1994a), and β-lactamase (Stemmer, 1994b). In DNA shuffling, genes are broken into small, random fragments with DNase I, and then reassembled in a PCR-like reaction, but without any primers. The process of reassembling can be mutagenic in the absence of a proofreading polymerase, generating up to 0.7% error when 10- to 50-bp fragments are used. These mutations consist of both transitions and transversion, randomly distributed over the length of the reassembled segment.

PCR-amplify the fragment to be shuffled. Often it is convenient to PCR from a bacterial colony or plaque. Touch the colony or plaque with a sterile toothpick and swirl in a standard PCR reaction mix (10 mM Tris-HC1, 50 mM KC1, 1.5 mM MgC12, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.005% Brij 35, 0.1 to 1 μM of each primer). Remove the toothpick and heat the reaction for 10 min at 99°C. Cool the reaction to 72°C, add 1–2 units of Taq DNA polymerase, cycle the reaction 35 times for 30 sec at 94°C, 30 sec at 45°C, 30 sec at 72°C, and finally heat the sample for 5 min at 72°C. (All the conditions here are for a 1-kb gene.)

Remove the free primers, preferably by purification of the DNA with Wizard PCR Preps (Promega, Madison, WI), or by gel purification. Complete primer removal is essential.

Fragment about 2–4 μg of the DNA with 0.15 units of DNase I (Sigma, St. Louis, MO) in 100 μl of 50 mM Tris·HC1 (pH 7.4), 1 mM MgC12, for 5–10 min at room temperature. Freeze on dry ice, check size range of fragments on 2% low-melting-point agarose gel or equivalent, and thaw to continue digestion until desired size range is obtained. The desired size range depends on the application; for shuffling of a 1-kb gene, fragments of 100–300 bases are adequate.

Gel purification. Gel-purify the desired DNA fragment size range (100–300 bp) from a 2% low-melting-point agarose gel or equivalent. One easy method is to insert a small piece of Whatman DE-81 ion-exchange paper just in front of the DNA, run the DNA into the paper, put the paper in 0.5 ml 1.2 M NaC1 in TE, vortex 30 sec, then carefully spin out all the paper, transfer the supernatant, and add 2 vol of 100% ethanol to precipitate the DNA; no cooling of the sample should be necessary. The DNA pellet is then washed with 70% ethanol to remove traces of salt.

DNA reassembly. The DNA pellet is resuspended in PCR mix (Promega) containing 0.2 mM each dNTP, 2.2 mM MgC12, 50 mM KC1, 10 mM Tris·HC1, pH 9.0, 0.1% Triton X-100, at a high concentration of 10–30 ng of fragments per microliter of PCR mix (typically 100–600 ng per 10–20 μl PCR reaction). No primers are added in this PCR reaction. Taq DNA polymerase (Promega) alone can be used if a substantial rate of mutagenesis (up to 0.7% with 10- to 50-bp DNA fragments) is desired. The inclusion of a proofreading polymerase, such as a 1/30 (vol/vol) mixture of Taq and Pfu DNA polymerase (Stratagene, San Diego, CA) is expected to yield a lower error rate (Barnes, 1994) and allows the PCR of very long sequences. A program of 30–45 cycles of 30 sec 94°C, 30 sec 45–50°C, 30 sec 72°C in an MJ Research PTC-150 minicycler (Cambridge, MA) is run. The progress of the assembly can be checked by gel analysis but this is normally not necessary. The PCR product at this point should contain the correct size product in a smear of larger and smaller sizes.

Amplification. The correctly reassembled product of this first PCR is amplified in a second PCR reaction which contains the outside primers. Aliquots of 2.5 μl of the PCR assembly are diluted 40x with PCR mix containing 0.8 μM of each primer. A PCR program of 20 cycles of 30 sec 94°C, 30 sec 50°C, and 30–45 sec at 72°C is run, with 5 min at 72°C at the end. This amplification results in a large amount of PCR product of the correct size.

Cloning. The best PCR product is then digested with terminal restriction enzymes, gel-purified, and cloned back into a phage or phagemid genome.

What are random changes in genetics called?

Is mutation a random change in DNA?