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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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Randomized primers. (a) Primers with random bases are inserted into the termini of the molecules by PCR and the reaction is terminated at the exponential phase to avoid hetero-dimers. (b) DNA molecules from the light green PCR shown in panel A are diluted and used as template for smPCR with the C–A primer (PCRs on single molecules). As control a ‘false positive’ smPCR with the same DNA but with many template molecules was also performed. (c) On the left: the sequencing chromatogram of the ‘false positive’ smPCR from panel b shows al 4 bases at the 3 random positions, indicating that the reaction was not a true smPCR. On the right: the sequencing chromatograms of four different smPCRs from panel B show only one base call at each of the three random positions, indicating they were true smPCRs.
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Figure 4: Randomized primers. (a) Primers with random bases are inserted into the termini of the molecules by PCR and the reaction is terminated at the exponential phase to avoid hetero-dimers. (b) DNA molecules from the light green PCR shown in panel A are diluted and used as template for smPCR with the C–A primer (PCRs on single molecules). As control a ‘false positive’ smPCR with the same DNA but with many template molecules was also performed. (c) On the left: the sequencing chromatogram of the ‘false positive’ smPCR from panel b shows al 4 bases at the 3 random positions, indicating that the reaction was not a true smPCR. On the right: the sequencing chromatograms of four different smPCRs from panel B show only one base call at each of the three random positions, indicating they were true smPCRs.

Mentions: To facilitate the simple identification of rare smPCRs that despite the measures reported above were still not performed on single molecules, we integrated another feature in our procedure, previously proposed for other smPCR applications (16). We incorporated oligos with three random bases at both ends of the synthetic DNA constructs that are to be cloned, effectively bar-coding the molecules with a four-letter code at six positions (46 = 4096 tags) (Figure 4a). Sequencing these molecules show that the sequence at the location of the random bases is always singular in the sequencing of a true smPCR (Figure 4d) and multiple in PCRs performed on >1 template molecules (Figure 4c).Figure 4.


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)

Randomized primers. (a) Primers with random bases are inserted into the termini of the molecules by PCR and the reaction is terminated at the exponential phase to avoid hetero-dimers. (b) DNA molecules from the light green PCR shown in panel A are diluted and used as template for smPCR with the C–A primer (PCRs on single molecules). As control a ‘false positive’ smPCR with the same DNA but with many template molecules was also performed. (c) On the left: the sequencing chromatogram of the ‘false positive’ smPCR from panel b shows al 4 bases at the 3 random positions, indicating that the reaction was not a true smPCR. On the right: the sequencing chromatograms of four different smPCRs from panel B show only one base call at each of the three random positions, indicating they were true smPCRs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Randomized primers. (a) Primers with random bases are inserted into the termini of the molecules by PCR and the reaction is terminated at the exponential phase to avoid hetero-dimers. (b) DNA molecules from the light green PCR shown in panel A are diluted and used as template for smPCR with the C–A primer (PCRs on single molecules). As control a ‘false positive’ smPCR with the same DNA but with many template molecules was also performed. (c) On the left: the sequencing chromatogram of the ‘false positive’ smPCR from panel b shows al 4 bases at the 3 random positions, indicating that the reaction was not a true smPCR. On the right: the sequencing chromatograms of four different smPCRs from panel B show only one base call at each of the three random positions, indicating they were true smPCRs.
Mentions: To facilitate the simple identification of rare smPCRs that despite the measures reported above were still not performed on single molecules, we integrated another feature in our procedure, previously proposed for other smPCR applications (16). We incorporated oligos with three random bases at both ends of the synthetic DNA constructs that are to be cloned, effectively bar-coding the molecules with a four-letter code at six positions (46 = 4096 tags) (Figure 4a). Sequencing these molecules show that the sequence at the location of the random bases is always singular in the sequencing of a true smPCR (Figure 4d) and multiple in PCRs performed on >1 template molecules (Figure 4c).Figure 4.

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