Limits...
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.

Show MeSH
Error-free molecules are readily cloned using smPCR. smPCR provides an alternative to in vivo cloning in de novo DNA synthesis up to the 2 Kb range at least. (a) For a 1-kb molecule and (b) for a 2-kb molecule show the probability that at least one of the molecules after error correction is error-free as a function of the number of molecules screened: blue plot—no error-correction or error-correction with smPCR using Taq (error-rate 1/200); green plot—error-correction with smPCR using a proofreading polymerase; red plot—error-correction with in vivo cloning. (c) The total (including clones of construction) number of clones needed for the construction of at least one error-free molecule with 90% probability as a function of the length of the molecule.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553596&req=5

Figure 5: Error-free molecules are readily cloned using smPCR. smPCR provides an alternative to in vivo cloning in de novo DNA synthesis up to the 2 Kb range at least. (a) For a 1-kb molecule and (b) for a 2-kb molecule show the probability that at least one of the molecules after error correction is error-free as a function of the number of molecules screened: blue plot—no error-correction or error-correction with smPCR using Taq (error-rate 1/200); green plot—error-correction with smPCR using a proofreading polymerase; red plot—error-correction with in vivo cloning. (c) The total (including clones of construction) number of clones needed for the construction of at least one error-free molecule with 90% probability as a function of the length of the molecule.

Mentions: Therefore, the entire procedure using Taq is noneffective for de novo DNA synthesis since the error-rate resulting from smPCR amplification is roughly the error-rate of the synthetic molecules before any error-correction. Moreover, error-correction using smPCR with Taq may even increase the number of clones needed compared to construction with no error-correction, depending on the error-rate of the oligos used (Figure 5c, dark blue and green plots).Figure 5.


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)

Error-free molecules are readily cloned using smPCR. smPCR provides an alternative to in vivo cloning in de novo DNA synthesis up to the 2 Kb range at least. (a) For a 1-kb molecule and (b) for a 2-kb molecule show the probability that at least one of the molecules after error correction is error-free as a function of the number of molecules screened: blue plot—no error-correction or error-correction with smPCR using Taq (error-rate 1/200); green plot—error-correction with smPCR using a proofreading polymerase; red plot—error-correction with in vivo cloning. (c) The total (including clones of construction) number of clones needed for the construction of at least one error-free molecule with 90% probability as a function of the length of the molecule.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553596&req=5

Figure 5: Error-free molecules are readily cloned using smPCR. smPCR provides an alternative to in vivo cloning in de novo DNA synthesis up to the 2 Kb range at least. (a) For a 1-kb molecule and (b) for a 2-kb molecule show the probability that at least one of the molecules after error correction is error-free as a function of the number of molecules screened: blue plot—no error-correction or error-correction with smPCR using Taq (error-rate 1/200); green plot—error-correction with smPCR using a proofreading polymerase; red plot—error-correction with in vivo cloning. (c) The total (including clones of construction) number of clones needed for the construction of at least one error-free molecule with 90% probability as a function of the length of the molecule.
Mentions: Therefore, the entire procedure using Taq is noneffective for de novo DNA synthesis since the error-rate resulting from smPCR amplification is roughly the error-rate of the synthetic molecules before any error-correction. Moreover, error-correction using smPCR with Taq may even increase the number of clones needed compared to construction with no error-correction, depending on the error-rate of the oligos used (Figure 5c, dark blue and green plots).Figure 5.

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