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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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Primer, dimers and anticipation. Adequate selection of primers leads to improved specificity in smPCR; RT-PCR can distinguish true smPCRs from false positives. (a) smPCRs with regular primers show many nonspecific amplification products. Top gel: Lanes 1–7: positive control (many template molecules) PCRs show bands at the correct size. Lanes 8–15: no-template control PCRs have nonspecific amplification from primers. Bottom gel: smPCR experiments—a large fraction of reactions show nonspecific amplification from primers which inhibit smPCR and hinder its use. (b) smPCRs with the C–A primer shows specific amplification. Top left gel: positive control (multiple template molecules) PCRs show bands at the correct size. Top right gel: no-template control PCRs do not have nonspecific amplification. Bottom gel: smPCR experiments bands at the correct size and frequency with no nonspecific amplification C. RT-PCR helps determining whether PCRs are true smPCRs or false positives due to nonspecific amplification from primers or contamination.
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Figure 2: Primer, dimers and anticipation. Adequate selection of primers leads to improved specificity in smPCR; RT-PCR can distinguish true smPCRs from false positives. (a) smPCRs with regular primers show many nonspecific amplification products. Top gel: Lanes 1–7: positive control (many template molecules) PCRs show bands at the correct size. Lanes 8–15: no-template control PCRs have nonspecific amplification from primers. Bottom gel: smPCR experiments—a large fraction of reactions show nonspecific amplification from primers which inhibit smPCR and hinder its use. (b) smPCRs with the C–A primer shows specific amplification. Top left gel: positive control (multiple template molecules) PCRs show bands at the correct size. Top right gel: no-template control PCRs do not have nonspecific amplification. Bottom gel: smPCR experiments bands at the correct size and frequency with no nonspecific amplification C. RT-PCR helps determining whether PCRs are true smPCRs or false positives due to nonspecific amplification from primers or contamination.

Mentions: smPCR amplification requires extensive cycling (9–12). This often leads to the amplification of nonspecific products originating from interaction between the PCR primers (Figure 2a). This often inhibits the amplification of the single molecule template, typically resulting in either no amplification of the target molecule due to dimer formation or in amplification of the primer dimer on top of the correct PCR product (Figure 2a). Consequently, a large fraction of the smPCRs performed cannot be used for synthesis since they did not amplify or have nonspecific amplification products. This has to be compensated for by performing more smPCRs than are actually needed for synthesis. To solve this problem we designed a special primer for smPCR consisting of a single sequence (complementary to both ends of the single molecule template), which contains a sequence of cytosine and adenine DNA bases only (see Supplementary Data for sequence). We reasoned that this should reduce the formation of PCR products that originate from primer-primer interactions due to the noncomplementary nature of the cytosine and adenine bases. This successfully eliminated nonspecific amplification resulting from interaction between primers and its inhibiting effect on single molecule amplification (Figure 2b), which in turn significantly decreased the total number of PCRs needed to obtain the minimal number of smPCR clones required for synthesis of error-free DNA. The sites for the C–A primer (as well as the random bar coding bases to be discussed later on) at the termini of the target molecules are incorporated by either an a priori PCR (16) or during the synthesis of the molecule as part of the target sequence.Figure 2.


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)

Primer, dimers and anticipation. Adequate selection of primers leads to improved specificity in smPCR; RT-PCR can distinguish true smPCRs from false positives. (a) smPCRs with regular primers show many nonspecific amplification products. Top gel: Lanes 1–7: positive control (many template molecules) PCRs show bands at the correct size. Lanes 8–15: no-template control PCRs have nonspecific amplification from primers. Bottom gel: smPCR experiments—a large fraction of reactions show nonspecific amplification from primers which inhibit smPCR and hinder its use. (b) smPCRs with the C–A primer shows specific amplification. Top left gel: positive control (multiple template molecules) PCRs show bands at the correct size. Top right gel: no-template control PCRs do not have nonspecific amplification. Bottom gel: smPCR experiments bands at the correct size and frequency with no nonspecific amplification C. RT-PCR helps determining whether PCRs are true smPCRs or false positives due to nonspecific amplification from primers or contamination.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Primer, dimers and anticipation. Adequate selection of primers leads to improved specificity in smPCR; RT-PCR can distinguish true smPCRs from false positives. (a) smPCRs with regular primers show many nonspecific amplification products. Top gel: Lanes 1–7: positive control (many template molecules) PCRs show bands at the correct size. Lanes 8–15: no-template control PCRs have nonspecific amplification from primers. Bottom gel: smPCR experiments—a large fraction of reactions show nonspecific amplification from primers which inhibit smPCR and hinder its use. (b) smPCRs with the C–A primer shows specific amplification. Top left gel: positive control (multiple template molecules) PCRs show bands at the correct size. Top right gel: no-template control PCRs do not have nonspecific amplification. Bottom gel: smPCR experiments bands at the correct size and frequency with no nonspecific amplification C. RT-PCR helps determining whether PCRs are true smPCRs or false positives due to nonspecific amplification from primers or contamination.
Mentions: smPCR amplification requires extensive cycling (9–12). This often leads to the amplification of nonspecific products originating from interaction between the PCR primers (Figure 2a). This often inhibits the amplification of the single molecule template, typically resulting in either no amplification of the target molecule due to dimer formation or in amplification of the primer dimer on top of the correct PCR product (Figure 2a). Consequently, a large fraction of the smPCRs performed cannot be used for synthesis since they did not amplify or have nonspecific amplification products. This has to be compensated for by performing more smPCRs than are actually needed for synthesis. To solve this problem we designed a special primer for smPCR consisting of a single sequence (complementary to both ends of the single molecule template), which contains a sequence of cytosine and adenine DNA bases only (see Supplementary Data for sequence). We reasoned that this should reduce the formation of PCR products that originate from primer-primer interactions due to the noncomplementary nature of the cytosine and adenine bases. This successfully eliminated nonspecific amplification resulting from interaction between primers and its inhibiting effect on single molecule amplification (Figure 2b), which in turn significantly decreased the total number of PCRs needed to obtain the minimal number of smPCR clones required for synthesis of error-free DNA. The sites for the C–A primer (as well as the random bar coding bases to be discussed later on) at the termini of the target molecules are incorporated by either an a priori PCR (16) or during the synthesis of the molecule as part of the target sequence.Figure 2.

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