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. 2007 Nov 14:7:77.
doi: 10.1186/1472-6750-7-77.

Revealing biases inherent in recombination protocols

Javier F Chaparro-Riggers  1 , Bernard Lw Loo, Karen M Polizzi, Phillip R Gibbs, Xiao-Song Tang, Mark J Nelson, Andreas S Bommarius
Affiliations

Revealing biases inherent in recombination protocols

Javier F Chaparro-Riggers et al. BMC Biotechnol. .

Abstract

Background: The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.

Results: We developed different test systems to compare and reveal biases from DNA shuffling and recombination-dependent PCR (RD-PCR), a StEP-like recombination protocol. An assay based on the reactivation of beta-lactamase was developed to simulate the recombination of point mutations. Both protocols performed similarly here, with slight advantages for RD-PCR. However, clear differences in the performance of the recombination protocols were observed when applied to homologous genes of varying DNA identities. Most importantly, the recombination-dependent PCR showed a less pronounced bias of the crossovers in regions with high sequence identity. We discovered that template variations, including engineered terminal truncations, have significant influence on the position of the crossovers in the recombination-dependent PCR. In comparison, DNA shuffling can produce higher crossover numbers, while the recombination-dependent PCR frequently results in one crossover. Lastly, DNA shuffling and recombination-dependent PCR both produce counter-productive variants such as parental sequences and have chimeras that are over-represented in a library, respectively. Lastly, only RD-PCR yielded chimeras in the low homology situation of GFP/mRFP (45% DNA identity level).

Conclusion: By comparing different recombination scenarios, this study expands on existing recombination knowledge and sheds new light on known biases, which should improve library-creation efforts. It could be shown that the recombination-dependent PCR is an easy to perform alternative to DNA shuffling.

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Figures

Figure 1
Figure 1
Recombination techniques used in this work. a) DNA Shuffling: Parental genes are randomly fragmented using DNaseI. The resulting fragments are recombined using a primer-free PCR using denaturation at high temperature, followed by annealing to other fragments, and extension by DNA polymerase. Some of these annealing events result in skew extension without recombination of fragments from two homologous parents, leading to parental background. After 35 cycles of assembly, PCR amplification with primers is used to selectively amplify full-length sequences. b) RD-PCR with one skew primer per parent: The templates are extended by parent-specific sequences resulting in asymmetric products by attaching distinct "head" and "tail". These sequences are used in the recombination PCR as primers to ensure crossover events. After the template denaturation, a high number of short annealing and extension steps results in template switching. Based on the asymmetric primers a complete product formation can only be amplified if an odd number of crossovers occurs. The resulting product will always contain different parents at the exposed ends. c) RD-PCR with two skew primers per parent: Parental templates are amplified with two unique skew primers in the first step (solid and dashed). The protocol then proceeds as in b) above, but the presence of the unique sequence prevents skew extension without recombination from happening.
Figure 2
Figure 2
Templates-pairs used for the recombination of mRFP (black) and DsRed (grey). Parental sequences or parental background are either mRFP WT or DsRed WT genes. Digested GFP pProTeT plasmids were used as cloning vectors for inserts to ensure that only fully cut plasmids were ligated with chimeric inserts.
Figure 3
Figure 3
Statistical analysis of the libraries generated by each protocol for DsRed/mRFP and GFP/mRFP. Useful sequences are sequences that would be interesting to screen. This measure excludes parental background and does not count duplicate sequences more than once. RD-PCRs 1–4 use one skew primer per parent (Figure 1b), RD-PCR 5 uses two skew primers per parent (Figure 1c). Templates (see Figure 2): RD-PCR1 and RD-PCR 5: full length DsRed/mRFP. RD-PCR 2: DsRed template with the first 5 bp truncated, full length mRFP. RD-PCR 3: DsRed template with 44 base pairs truncated from the 5' end, full length mRFP. RD-PCR 4: Both templates are truncated. RD-PCR 5: with two skew primers per parent.
Figure 4
Figure 4
Combined plot of the frequency and location of crossovers in libraries made from DsRed and mRFP. Shading indicates that bases are identical in DsRed and mRFP; white space indicates that bases differ. Crossovers are denoted at the position where the first base pair differs between the two sequences. Sequences with multiple crossovers were marked at each crossover position separately. 'One skew primer per parent' combines the results from RD-PCR1-4. Subplots of Figure 4 can be found in additional file 2.
Figure 5
Figure 5
Local identity required for a crossover to occur. The highest number of continuous identical base pairs on either side of the crossover region is plotted versus the percentage of crossovers that contain that number. The DNA shuffling distribution differs significantly from the RD-PCR distribution as determined by the Wilcoxon Rank-Sum test (normal approximation to determine the p-value, p = 0.028).
Figure 6
Figure 6
Frequency and location of crossovers obtained from recombination of HcRed and DsRed. (a): The frequency and location of crossovers in DNA shuffled libraries obtained from HcRed and DsRed. The lines indicate rolling DNA identity calculated by summing the number of identical DNA bp in a 20 bp window and dividing by 20 bp. So, one would indicate that 100% DNA identity in a 20 bp window, ten to the left and ten to the right of a DNA residue. (b): The frequency and location of crossovers in RD-PCR libraries obtained from HcRed and DsRed.
Figure 7
Figure 7
The frequency and location of crossovers in libraries made from GFP and mRFP. Shading indicates that bases are identical in GFP and mRFP; white space indicates that bases differ. Since the genes are of different length, gaps in the mRFP sequence were excluded.
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