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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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Overview—Although de novo DNA synthesis is traditionally performed with in vivo cloning, which is time consuming and labor intensive, it can, in principle, be performed instead in vitro using a modified smPCR protocol. (a) Work reported here: Target synthetic molecules are recursively constructed (5) from oligos and then error-corrected using the new smPCR procedure instead of in vivo cloning. In brief, Preparation of the target DNA molecules for smPCR amplification is carried out by a PCR that introduces sites for the smPCR primer (see text). This PCR is stopped at the exponential phase of amplification so that heterodimers are not formed (see text). The PCR products are then diluted according to calculations and experimental results (see text) and used as template for smPCR with a special primer (C–A primer) that doesn't produce nonspecific amplification products (see text). The DNA ‘clones’ amplified using smPCR are then sequenced and an error-correction process (5) (Also see Supplementary Data Methods section for error-correction description) is carried out using the smPCR amplified molecules as starting material until an error free molecule is obtained. (b) Conceptual illustration of how the smPCR procedure could also be used in principle, with a two-step assembly PCR. From left to right, oligos are assembled in groups and amplified to yield fragments 400–500 bp long. These could be cloned using exactly the same smPCR procedure described in this work and sequenced. The error-free clones are then selected for further assembly of the target sequence using various methodologies.
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Figure 1: Overview—Although de novo DNA synthesis is traditionally performed with in vivo cloning, which is time consuming and labor intensive, it can, in principle, be performed instead in vitro using a modified smPCR protocol. (a) Work reported here: Target synthetic molecules are recursively constructed (5) from oligos and then error-corrected using the new smPCR procedure instead of in vivo cloning. In brief, Preparation of the target DNA molecules for smPCR amplification is carried out by a PCR that introduces sites for the smPCR primer (see text). This PCR is stopped at the exponential phase of amplification so that heterodimers are not formed (see text). The PCR products are then diluted according to calculations and experimental results (see text) and used as template for smPCR with a special primer (C–A primer) that doesn't produce nonspecific amplification products (see text). The DNA ‘clones’ amplified using smPCR are then sequenced and an error-correction process (5) (Also see Supplementary Data Methods section for error-correction description) is carried out using the smPCR amplified molecules as starting material until an error free molecule is obtained. (b) Conceptual illustration of how the smPCR procedure could also be used in principle, with a two-step assembly PCR. From left to right, oligos are assembled in groups and amplified to yield fragments 400–500 bp long. These could be cloned using exactly the same smPCR procedure described in this work and sequenced. The error-free clones are then selected for further assembly of the target sequence using various methodologies.

Mentions: We expect that our methods may be used to incorporate smPCR also in other DNA synthesis procedures, for example in conjunction with the widely used two step assembly PCR method (7) (Figure 1b).Figure 1.


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)

Overview—Although de novo DNA synthesis is traditionally performed with in vivo cloning, which is time consuming and labor intensive, it can, in principle, be performed instead in vitro using a modified smPCR protocol. (a) Work reported here: Target synthetic molecules are recursively constructed (5) from oligos and then error-corrected using the new smPCR procedure instead of in vivo cloning. In brief, Preparation of the target DNA molecules for smPCR amplification is carried out by a PCR that introduces sites for the smPCR primer (see text). This PCR is stopped at the exponential phase of amplification so that heterodimers are not formed (see text). The PCR products are then diluted according to calculations and experimental results (see text) and used as template for smPCR with a special primer (C–A primer) that doesn't produce nonspecific amplification products (see text). The DNA ‘clones’ amplified using smPCR are then sequenced and an error-correction process (5) (Also see Supplementary Data Methods section for error-correction description) is carried out using the smPCR amplified molecules as starting material until an error free molecule is obtained. (b) Conceptual illustration of how the smPCR procedure could also be used in principle, with a two-step assembly PCR. From left to right, oligos are assembled in groups and amplified to yield fragments 400–500 bp long. These could be cloned using exactly the same smPCR procedure described in this work and sequenced. The error-free clones are then selected for further assembly of the target sequence using various methodologies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Overview—Although de novo DNA synthesis is traditionally performed with in vivo cloning, which is time consuming and labor intensive, it can, in principle, be performed instead in vitro using a modified smPCR protocol. (a) Work reported here: Target synthetic molecules are recursively constructed (5) from oligos and then error-corrected using the new smPCR procedure instead of in vivo cloning. In brief, Preparation of the target DNA molecules for smPCR amplification is carried out by a PCR that introduces sites for the smPCR primer (see text). This PCR is stopped at the exponential phase of amplification so that heterodimers are not formed (see text). The PCR products are then diluted according to calculations and experimental results (see text) and used as template for smPCR with a special primer (C–A primer) that doesn't produce nonspecific amplification products (see text). The DNA ‘clones’ amplified using smPCR are then sequenced and an error-correction process (5) (Also see Supplementary Data Methods section for error-correction description) is carried out using the smPCR amplified molecules as starting material until an error free molecule is obtained. (b) Conceptual illustration of how the smPCR procedure could also be used in principle, with a two-step assembly PCR. From left to right, oligos are assembled in groups and amplified to yield fragments 400–500 bp long. These could be cloned using exactly the same smPCR procedure described in this work and sequenced. The error-free clones are then selected for further assembly of the target sequence using various methodologies.
Mentions: We expect that our methods may be used to incorporate smPCR also in other DNA synthesis procedures, for example in conjunction with the widely used two step assembly PCR method (7) (Figure 1b).Figure 1.

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