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Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis

By Hongye Ye, Mo Chao Huang, Mo-Huang Li and Jackie Y. Ying

Abstract

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10°C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis

Topics: Methods Online
Publisher: Oxford University Press
OAI identifier: oai:pubmedcentral.nih.gov:2673447
Provided by: PubMed Central
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    Citations

    1. (2008). A modified method for PCR-directed gene synthesis from large number of overlapping oligodeoxyribonucleotides.
    2. (2004). A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences.
    3. (2006). Amplicon DNA melting analysis for mutation scanning and genotyping: Cross-platform comparison of instruments and dyes.
    4. (2003). Amplification efficiency of thermostable DNA polymerase.
    5. (2002). Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template.
    6. (2008). Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.
    7. (1997). Continuous fluorescence monitoring of rapid cycle DNA amplification.
    8. (2005). Correcting errors in synthetic DNA through consensus shuffling.
    9. (2003). Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR.
    10. (2006). Disposable real-time microPCR device: Lab-on-a-chip at a low cost.
    11. (2002). DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis.
    12. (1992). Dual asymmetric PCR: One-step construction of synthetic genes.
    13. (2005). Efficient initial pool generation for weighted graph problems using parallel overlap assembly.
    14. (1991). Excessive cycling converts PCR products to random-length higher molecular weight fragments.
    15. (1998). Gene synthesis by a LCR-based approach: High-level production of leptin-L54 using synthetic gene in Escherichia coli.
    16. (2003). Generating a synthetic genome by whole genome assembly: X174 bacteriophage from synthetic oligonucleotides.
    17. (2003). High-resolution genotyping by amplicon melting analysis using LCGreen.
    18. (1998). Influence of magnesium ion concentration and PCR amplification conditions on cross-species PCR.
    19. (2009). Integrated two-step gene synthesis in a microfluidic device.
    20. (2001). Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg 2+, deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas.
    21. (2007). Optimal encoding rules for synthetic genes: the need for a community effort.
    22. (2007). Parallel gene synthesis in a microfluidic device.
    23. (2007). Partial strands synthesizing leads to inevitable aborting and complicated products in consecutive polymerase chain reactions (PCRs).
    24. (2006). PCR-based accurate synthesis of long DNA sequences.
    25. (2007). Protein fabrication automation.
    26. (2004). Protein-mediated error correction for de novo DNA synthesis.
    27. (2007). Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes.
    28. (2006). Real-time PCR and SYBR Green I melting curve analysis for the identification of plum pox virus strains C, EA, and W: effect of amplicon size, melt rate, and dye translocation.
    29. (2005). Reconstruction of genetic circuits.
    30. (1992). Recursive PCR: A novel technique for total gene synthesis.
    31. (2005). Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage.
    32. (2007). S100A4 accelerates tumorigenesis and invasion of human prostate cancer through the transcriptional regulation of matrix metalloproteinase 9.
    33. (2004). Self-assembling protein microarrays.
    34. (2006). Simplified gene synthesis: A one-step approach to PCR-based gene construction.
    35. (1995). Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides.
    36. (1999). Statistical mechanical simulation of polymeric DNA melting with MELTSIM.
    37. (2004). The thermodynamics of DNA structural motifs.
    38. (2003). Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: A novel method of primer design for highfidelity assembly of longer gene sequences.
    39. (2004). Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster.
    40. (1991). Touchdown’ PCR to circumvent spurious priming during gene amplification.
    41. (2004). Two-step total gene synthesis method.
    42. (2007). Use of DNA melting simulation software for in silico diagnostic assay design: Targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes.

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