Staggered Extension Process In Vitro DNA Recombination

Introduction

Directing the evolution of proteins and nucleic acids for any of our purpose can be obtained by employing in vitro DNA recombination method. This method is an extremely efficient and powerful method.  There are random mutagenesis methods which employ point mutations. These point mutations are introduced randomly into a single parent sequence. Thus the result will produce a library of progeny sequences. Unlike these methods, DNA recombination methods involve the block-wise exchange of genetic variations. These variations can be carried out among multiple parent sequences.

The parent sequences may be created in the laboratory or they exist in nature. The end product of this process is a library of chimeric progeny sequences. When a mutation turns out to be beneficial it is accumulated while deleterious mutations are removed by this method. This leads to a fast-evolving protein or nucleic acid molecule. This evolution moves towards a specific function. The first demonstration of the role of DNA recombination in the evolution of biological systems is done using a computer simulation. In vitro DNA recombination has found its application to alter and engineer many types of protein function.

It has been used to change the following attributes of the protein: stability, activity, affinity, selectivity, substrate specificity, and protein folding/solubility. DNA shuffling is an in vitro DNA recombination method. DNA fragments generated by the random digestion of parent genes with DNase I are combined and reassembled into full-length chimeric progeny genes in a polymerase chain reaction (PCR)-like process. A list of various in-vitro DNA recombination has been studied. This includes methods such as Staggered extension process (StEP) recombination, random-priming recombination (RPR), random chimeragenesis on transient templates (RACHITT), degenerate homoduplex recombination (DHR) and synthetic shuffling.

The implication of the use of DNA recombination over random mutagenesis has been demonstrated using the molecular evolution of TEM-1 b-lactamase.  The DNA recombination in this study implies the usage of three rounds of DNA shuffling and it is followed by two rounds of backcrossing with the wild-type gene resulted in mutants. This directed evolution resulted in 32000-fold improvement in the minimum inhibitory concentration (MIC) of the antibiotic cefotaxime. A PCR method is used in this application as control/ comparative study yielded only a 16-fold improvement in MIC after three iterations.

In this article, method, protocol, and applications of StEP recombination are described.

Principle of the StEP Method

In a PCR process, during the polymerase-catalyzed primer extension step, switching of the primers leads to StEP recombination in a DNA.  Full-length genes are used as templates for the synthesis of chimeric progeny genes. StEP includes priming of denatured templates, followed by repeated cycles of denaturation and extremely short annealing/extension steps. The recombination occurs due to random annealing of the partially extended primers to different templates. This step is based on sequence complementarity. This process continues until full-length genes are formed. In case of low product yield, standard PCR methods can be used to amplify the full-length chimeric genes. Due to the independence of the fragmentation step (which is present in other recombination methods) the StEP method is much simpler and less labor intensive. StEP can be performed using a pair of flanking primers in a PCR tube. StEP mimics the process that retroviruses use to evolve their genomes.

Methodology

A green fluorescent protein (GFP)-based recombination test system is used to test the recombination efficiency of the StEP method was compared to the most widely used DNA shuffling method. For this test, a series of truncated GFP variants containing stop codon mutations that are nonfluorescent were created by site-directed mutagenesis at selected positions along the GFP gene was used.  The recombination that is happening between truncated GFP variants generates the full-length wild-type gene and thus restores the fluorescence property of the protein.  The recombination frequency or efficiency between two stop codon mutations of a given distance is expressed as the percentage of fluorescent host Escherichia coli colonies.

Materials

  • DNA templates containing the target sequences to be recombined Oligonucleotide primers
  • Taq DNA polymerase and its 10 X reaction buffer:
  • 500 mM KCl,
  • 100 mM Tris–HCl, pH 8.3
  • 25 mM MgCl2
  • 10 X dNTP mix: 2 mM of each dNTP
  • Agarose gel electrophoresis supplies and equipment
  • Thermocycler
  • Gel extraction kit
  • DpnI restriction endonuclease (20 U/ml)
  • 10 X supplied reaction buffer

Experimental Approach

  1.  Prepare DNA template. Appropriate templates include plasmids carrying target sequences, cDNA or genomic DNA carrying the target sequences, sequences excised by restriction endonucleases, and PCRamplified sequences.
  2. Combine 5 ml of 10 X Taq buffer, 5 ml of 10X dNTP mix (2 mM of each dNTP), 1.5 mM MgCl2, 1–20 ng total template DNA, 30–50 pmol of each primer, sterile dH2O, and 2.5 U Taq DNA polymerase in a total volume of 50 m
  3. Run 80–100 extension cycles using the following program: 940 for 30 s (denaturation) and 550 for 5–15 s (annealing/extension).
  4. Run a small aliquot (5–10 ml) of the reaction on an agarose gel. Possible reaction products are full-length amplified sequences, a smear, or a combination of both. If a discrete band with sufficient yield for subsequent cloning is obtained after the StEP reaction, no additional amplification step is needed. Proceed to step 8.
  5. (Optional) If parent templates were isolated from a dam methylation-positive E. coli strain (e.g., DH5a, XL1-Blue), the products from extension reactions can be incubated with DpnI endonuclease to remove parent DNA so as to reduce the background of nonchimeric genes. Combine 2 ml of the StEP reaction, 1X DpnI reaction buffer, 6 ml of sterile dH2O, and 1 ml of DpnI restriction endonuclease. Incubate at 370 for 1 h.
  6. Amplify the target sequence in a standard PCR. Combine 1ml of the StEP reaction, 0.3–1.0 mM of each primer, 10 ml of 10X Taq buffer, 1.5 mM MgCl2, 10 ml of 10X dNTP mix (2 mM of each dNTP), and 2.5 U Taq DNA polymerase in a total volume of 100 m Run the PCR reaction using the following program: 960 for 2 min, 25 cycles of 30 s at 940, 30 s at 550, and 60 s at 720 for each 1 kb in length. The final step of elongation is at 720 for 7 min.
  7. Run a small aliquot of the reaction mixture (5–10 ml) on an agarose gel. In most cases, a clear, discrete band of the correct size among a smear should be obtained.
  8. Purify the product of correct size using the gel purification kit. Digest the product with the appropriate restriction endonucleases and ligate into the desired cloning vector.

Notes

  1. Primer design.

Primer design should follow standard criteria, including similar melting temperatures and elimination of self-complementarity or complementarity of primers to each other. Free computer programs such as Primer3 at Biology Workbench can be used to design primers. Typically, primers should also include unique restriction sites for subsequent directional subcloning.

  1. Choice of a DNA polymerase.

The key to successful recombination by StEP is to tightly control the polymerase-catalyzed DNA extension. Too much extension during each StEP cycle will severely limit recombination events. Thermostable DNA polymerases currently used in DNA amplification are often very fast. Even very brief cycles of denaturation and annealing provide time for these enzymes to extend primers for hundreds of nucleotides. For example, extension rates of Taq DNA polymerase at various temperatures are: 700, >60 nucleotides/s; 550, ~24 nucleotides/s; 370, ~1.5 nucleotides/s; 220, ~0.25 nucleotides/s. Thus, it is not unusual for the full-length gene product to appear after only 10–15 cycles. Unfortunately, the faster the full-length gene product appears in the extension reaction, the lower the recombination frequency due to the fewer number of the template switching events. To increase the recombination frequency, various measures should be taken to minimize the time spent in each StEP cycle, including selecting a faster thermocycler, reducing the reaction volume, and using smaller PCR tubes with thin walls.

Alternatively, thermostable DNA polymerases with proofreading activity can be used. It was reported that the proofreading activity of high fidelity DNA polymerases can significantly slow down their extension rates. For example, Vent DNA polymerase has an extension rate of 1000 nucleotides/min and a processivity of 7 nucleotides/(initiation event) as compared to >4000 nucleotides/min and 40 nucleotides/(initiation event) for Taq DNA polymerase at a certain extension temperature. In addition, use of these alternative polymerases is highly recommended during DNA amplification to minimize the mutagenic rate of point mutations. Commercially available thermostable DNA polymerases with proofreading activity include Pfu DNA polymerase, and Pfx DNA polymerase. When setting up reactions with these polymerases, it is very important to add the polymerase last, as in the absence of dNTPs, the 30 to 50 exonuclease activity of the polymerase can degrade DNAs.

  1. Choice of annealing/extension temperatures and times.

As a general rule, the annealing temperature should be a few degrees lower than the melting temperature of the primers. The annealing temperature should be decreased when higher recombination frequency is required or when templates have low GC content. However, it should not be reduced too much in order to minimize nonspecific annealing events. The annealing/ extension times are chosen based on the desired recombination frequency. Both shorter extension times and lower annealing temperatures will increase the recombination frequency. The number of the annealing/ extension cycles are determined by the size of the full-length gene product.

  1. Extension products.

The progress of the StEP reaction can be monitored by taking aliquots of the reaction mixture at various time points and separating the DNA fragments by agarose gel electrophoresis. The appearance of the extension products may depend on the specific sequences recombined or the type of templates used. Small templates will likely show gradual accumulation of the full-length gene products with an increasing number of cycles. For example, during StEP recombination of two subtilisin E genes (~1 kb), the average size of the extension products increases gradually with increasing cycle number: 100 bp after 20 cycles, 400 bp after 40 cycles, 800 bp after 60 cycles, and a clear discrete band around 1 kb (the desired size) after 80 cycles. However, using large templates such as whole plasmids and long genes may result in nonspecific annealing of primers and their extension products throughout the templates. Although it may appear as a smear on the agarose gel, the increase of the size of their extension products may not be so obvious.

  1. PCR amplification.

If the PCR amplification reaction is not successful, i.e., no discrete band with sufficient yield is produced, repeat amplification using serial dilutions of the StEP reaction mixture: 1:10 dilution, 1:20 dilution, and 1:50 dilution. Run small aliquots of the amplified products on an agarose gel to determine the yield and quality of amplification. Select the reaction with a higher yield and lower amount of nonspecific products for subsequent cloning. An alternative solution is to use nested internal primers separated by 50–100 bp from the original primers to amplify the target sequences.

Applications of the StEP Method

StEP method can be successfully applied for most cases of recombination of gene variants. This method can be applied irrespective of the origin of the gene variants (which may occur due to random mutagenesis or naturally occurring homologous genes that are approximately 80% identical). One of its application is used to increase the temperature optimum of subtilisin E by 180 over that of the wild-type enzyme. This use of StEP converted a mesophilic enzyme into its thermophilic.  This technique has also been used for broadening the substrate specificity of biphenyl dioxygenase. It is done by the recombination of two homologous bphA genes encoding Burkholderia cepacia LB400 biphenyl dioxygenase and Pseudomonas pseudoalcaligenes KF707 biphenyl dioxygenase, respectively. The resultant product has the capability of degrading both congeners of ortho-(LB400) or para-(KF707) substituted polychlorinated biphenyls, instead of anyone as in case of their parental genes.

In certain cases a combination of  StEP recombination and DNA shuffling has been used. It is used to direct evolution or for construction of structure-based rational design of enzymes.  A novel function in an a/b barrel enzyme is achieved. This is done by converting the activity of indole-3-glycerol–phosphate synthase (IGPS) to that of phosphoribosyl anthranilate isomerase (PRAI). This incorporates a structure-based design that modifies the IGPS a/b barrel. This is achieved by the inclusion of the basic design of the loop system of PRAI, yielding a chimeric variant with very low PRAI activity.  After this, the recombination methods are used to enrich the PRAI activity. This enrichment results in a creation of a variant whose activity is six-fold higher than the wild-PRAI and exhibits no IGPS activity.

The comparison of the StEP method and DNA shuffling is done using two thermostable subtilisin E mutants. The resultant mutants showed similar DNA recombination efficiency in both the methods. Also in these methods, a minimum of one to four crossovers was observed.  The advantage of the StEP method is that the intrinsic ability of PCR to generate chimeric progeny genes from mixed homologous templates. As discussed previously this homologous recombination tactic is similar to that of the retroviruses such as HIV naturally use to evolve their genomes.

StEP recombination has been applied in few other applications, such as regioselectivity of a Bacillus a-galactosidase,  thermostabilization of cellulosomal endoglucanase EngB (this is done by recombining its gene with the homologus gene encoding the noncellulosomal endoglucanase EngD), and in increasing the protein expression level and enzyme activity of fugal laccase in Saccharomyces cerevisiae.

StEP method and random mutagenesis have also been used in combination with a certain application. This combined application has been used to convert cytochrome P450 BM-3 from Bacillus megaterium into a soluble, self-sufficient, highly active alkane hydroxylase. When the StEP procedure is coupled with a compartmentalized self-replication (CSR)-based high-throughput screening method, it has been used to significantly expand the substrate spectrum of Taq DNA polymerase for biotechnological applications. This combination was used to produce highly fluorescent microarray or in situ hybridization (FISH) probes.  Also in recent times, the StEP method was applied to aid the evolution of adeno-associated virus to exhibit enhanced properties such as a better ability to evade antibody neutralization for gene delivery.