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De novo DNA synthesis using single molecule PCR.

Ben Yehezkel T, Linshiz G, Buaron H, Kaplan S, Shabi U, Shapiro E - Nucleic Acids Res. (2008)

Bottom Line: The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification.The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck.Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.

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Heterodimers hinder smPCR. The template for smPCR is produced with an ordinary PCR reaction. If this PCR is not not terminated at the exponential phase of amplification it produces heterodimers, which hinder smPCR. (a) Overcycling of the PCR past the exponential phase of amplification leads to the formation of hetero-dimers by re-annealing of different elongated strands. (b) The sequencing chromatograms of both sense and antisense strands of a PCR amplified heterodimer are frame-shifted and unreadable from the site of the (insertion or deletion) mutation and on. (c) A PCR terminated before the end of the exponential amplification generates homodimers, not heterodimers. (d) The sequencing chromatogram of a PCR amplified homodimer is readable and not frame-shifted even if a mutations (with respect to the target sequence) are present.
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Figure 3: Heterodimers hinder smPCR. The template for smPCR is produced with an ordinary PCR reaction. If this PCR is not not terminated at the exponential phase of amplification it produces heterodimers, which hinder smPCR. (a) Overcycling of the PCR past the exponential phase of amplification leads to the formation of hetero-dimers by re-annealing of different elongated strands. (b) The sequencing chromatograms of both sense and antisense strands of a PCR amplified heterodimer are frame-shifted and unreadable from the site of the (insertion or deletion) mutation and on. (c) A PCR terminated before the end of the exponential amplification generates homodimers, not heterodimers. (d) The sequencing chromatogram of a PCR amplified homodimer is readable and not frame-shifted even if a mutations (with respect to the target sequence) are present.

Mentions: smPCR reactions are generally similar to regular PCR reactions in their basic biochemistry, the difference is that while PCR typically start the amplification with multiple copies of the template molecule, the goal in smPCR is to amplify a single template molecule. This is achieved by diluting a solution with template molecules in a known concentration so that the template aliquot is expected to have about one molecule. As the dilution is a stochastic process, at any such dilution some aliquots would have no template molecule and some would have multiple template molecules. As these two cases cannot be avoided, smPCR is done as a batch of multiple parallel reactions, with the hope that at least some would be true smPCRs, namely successful PCR reactions that amplify single template molecules. ‘False positive’ smPCRs, which amplify multiple template molecules, are identified using sequencing (Figure 3 and Supplementary Figure 6). The cost of sequencing is a major component of synthetic DNA synthesis, and the sequencing of false positives can render smPCR unpractical if their fraction in the total number of reactions is too high. Standard gel/capillary electrophoreses (CE)/RT-PCR analyses can be used to differentiate no template (negative) reactions from (positive) PCRs with template, however, they cannot be used to differentiate a true smPCR from false positive reactions. Diluting the template to one molecule per well on average maximizes the fraction of true smPCRs out of all the reactions in the batch (Supplementary Figure 3a, blue plot). However, it does not maximize the ratio of true smPCRs to false positives (Supplementary Figure 3a, green plot) which is important for avoiding futile sequencing. For example, aiming for one molecule per well on average leads to >50% futile sequencing of false positives (Supplementary Figure 3a, green plot). Further reducing template concentration reduces the extent of futile sequencing of PCRs with multiple template molecules, however, it increases the extent of futile PCRs due to no template reactions. Determining the template concentration that would result in an optimal ratio between true smPCRs, false positives and no template reactions can only be determined by associating a cost to performing sequencing and smPCR reactions. We calculated the optimal concentration to be ∼0.6 template molecules per smPCR well if an equal cost is associated with smPCR and sequencing (Supplementary Figure 3b), and ∼0.2 molecules per well if sequencing is assigned the more realistic cost of eight times that of smPCR (Supplementary Figure 3c). Performing smPCRs at the optimal template concentration reduces the overall cost of obtaining each sequenced true smPCR and the overall cost of using smPCR with de novo DNA synthesis since it reduces futile sequencing from 50% (with 1 molecule/well) to 10% (with ∼0.2 molecules/well) (Supplementary Figure 3a). A standard 260 nm OD measurement can be used to determine the optimal concentration.Figure 3.


De novo DNA synthesis using single molecule PCR.

Ben Yehezkel T, Linshiz G, Buaron H, Kaplan S, Shabi U, Shapiro E - Nucleic Acids Res. (2008)

Heterodimers hinder smPCR. The template for smPCR is produced with an ordinary PCR reaction. If this PCR is not not terminated at the exponential phase of amplification it produces heterodimers, which hinder smPCR. (a) Overcycling of the PCR past the exponential phase of amplification leads to the formation of hetero-dimers by re-annealing of different elongated strands. (b) The sequencing chromatograms of both sense and antisense strands of a PCR amplified heterodimer are frame-shifted and unreadable from the site of the (insertion or deletion) mutation and on. (c) A PCR terminated before the end of the exponential amplification generates homodimers, not heterodimers. (d) The sequencing chromatogram of a PCR amplified homodimer is readable and not frame-shifted even if a mutations (with respect to the target sequence) are present.
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Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Heterodimers hinder smPCR. The template for smPCR is produced with an ordinary PCR reaction. If this PCR is not not terminated at the exponential phase of amplification it produces heterodimers, which hinder smPCR. (a) Overcycling of the PCR past the exponential phase of amplification leads to the formation of hetero-dimers by re-annealing of different elongated strands. (b) The sequencing chromatograms of both sense and antisense strands of a PCR amplified heterodimer are frame-shifted and unreadable from the site of the (insertion or deletion) mutation and on. (c) A PCR terminated before the end of the exponential amplification generates homodimers, not heterodimers. (d) The sequencing chromatogram of a PCR amplified homodimer is readable and not frame-shifted even if a mutations (with respect to the target sequence) are present.
Mentions: smPCR reactions are generally similar to regular PCR reactions in their basic biochemistry, the difference is that while PCR typically start the amplification with multiple copies of the template molecule, the goal in smPCR is to amplify a single template molecule. This is achieved by diluting a solution with template molecules in a known concentration so that the template aliquot is expected to have about one molecule. As the dilution is a stochastic process, at any such dilution some aliquots would have no template molecule and some would have multiple template molecules. As these two cases cannot be avoided, smPCR is done as a batch of multiple parallel reactions, with the hope that at least some would be true smPCRs, namely successful PCR reactions that amplify single template molecules. ‘False positive’ smPCRs, which amplify multiple template molecules, are identified using sequencing (Figure 3 and Supplementary Figure 6). The cost of sequencing is a major component of synthetic DNA synthesis, and the sequencing of false positives can render smPCR unpractical if their fraction in the total number of reactions is too high. Standard gel/capillary electrophoreses (CE)/RT-PCR analyses can be used to differentiate no template (negative) reactions from (positive) PCRs with template, however, they cannot be used to differentiate a true smPCR from false positive reactions. Diluting the template to one molecule per well on average maximizes the fraction of true smPCRs out of all the reactions in the batch (Supplementary Figure 3a, blue plot). However, it does not maximize the ratio of true smPCRs to false positives (Supplementary Figure 3a, green plot) which is important for avoiding futile sequencing. For example, aiming for one molecule per well on average leads to >50% futile sequencing of false positives (Supplementary Figure 3a, green plot). Further reducing template concentration reduces the extent of futile sequencing of PCRs with multiple template molecules, however, it increases the extent of futile PCRs due to no template reactions. Determining the template concentration that would result in an optimal ratio between true smPCRs, false positives and no template reactions can only be determined by associating a cost to performing sequencing and smPCR reactions. We calculated the optimal concentration to be ∼0.6 template molecules per smPCR well if an equal cost is associated with smPCR and sequencing (Supplementary Figure 3b), and ∼0.2 molecules per well if sequencing is assigned the more realistic cost of eight times that of smPCR (Supplementary Figure 3c). Performing smPCRs at the optimal template concentration reduces the overall cost of obtaining each sequenced true smPCR and the overall cost of using smPCR with de novo DNA synthesis since it reduces futile sequencing from 50% (with 1 molecule/well) to 10% (with ∼0.2 molecules/well) (Supplementary Figure 3a). A standard 260 nm OD measurement can be used to determine the optimal concentration.Figure 3.

Bottom Line: The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification.The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck.Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.

Show MeSH