Oligonucleotide Stitching
The unique oligonucleotide stitching feature of the GeneArt® High-Order Genetic Assembly System uses short synthetic linkers to bridge the ends of existing DNA fragments, enabling the precise assembly of unrelated DNA fragments that do not share end-terminal homology without the need for PCR.
Figure 1: Cloning 2 DNA fragments in the pYES1L vector.
In the first two examples (Figure 1), the cloning efficiencies for two different oligonucleotide stitching experiments are shown using pYES1L as the vector. The procedure can also be used to introduce deletions, insertions, or other desired modifications to the final assembled recombinant DNA molecule.
How it works
GeneArt® High-Order and Genetic Assembly relies on yeast’s ability to take up and recombine DNA fragments with high efficiency. This process, termed transformation-associated recombination, greatly reduces handling of DNA and eliminates restriction endonuclease digestion and ligation while allowing precise assembly of DNA fragments.
GeneArt® Seamless Cloning and Assembly Kits work great together with GeneArt® Strings™ DNA Fragments. This new service offers custom-made DNA fragments up to 1,000 bp ready for cloning and assembly and delivered in only
Figure 2 The system is used to assemble three DNA fragments, one of which has end-terminal homology with the linear cloning vector. The remaining fragments are assembled using stitching oligonucleotides.
After verifying “positive” colonies containing the final assembled construct, the construct is transferred into E. coli for large scale plasmid preps for downstream applications.
High-Order DNA Assembly Protocol
- Combine the linear cloning vector (pYES1L or your own yeast-adapted vector) and the DNA fragments you want to assemble in a centrifuge tube. If you are assembling non-homologous DNA fragments, also include the stitching oligonucleotides in the same tube.
We highly recommend using our web-based tool to confirm end-terminal homology between adjacent DNA fragments, check for potential homologous regions in the fragments that could lead to undesired recombination, and/or to design PCR primers and stitching oligonucleotides. - Co-transform the linear cloning vector and the DNA fragments into MaV203 Competent Yeast Cells (supplied with the system). Plate the transformed yeast cells onto CSM-Trp agar plates and incubate for three days.
- Perform yeast colony PCR to screen positive yeast colonies containing your assembled recombinant DNA molecule. Isolate one or more of the positive colonies to transfer the assembled DNA molecule into E. coli.
- Lyse the positive yeast colony with the proprietary buffer and beads included in the system and use the lysate to transform the assembled DNA molecule into One Shot® TOP10 Electrocomp™ E. coli (supplied with the system) for large scale plasmid prep for downstream applications.
Unprecedented Precision and Efficiency
The GeneArt® High-Order Genetic Assembly System provides high cloning efficiency across a wide range of fragment sizes and numbers and enables the seamless assembly of DNA fragments precisely and without extra sequences.
Figure 3: Cloning efficiency for different numbers of 10 kbp fragments amplified by PCR.
Figure 3 illustrates the high cloning efficiency for 1, 3 and 10 DNA fragments of 10 kbp each, amplified by Platinum® PCR SuperMix High Fidelity and cloned into pYES1L. Constructs were transferred to E. coli and the DNA junctions were sequenced for accuracy.
Figure 4: Comparison of the cloning efficiencies of the GeneArt® Seamless Cloning and Assembly Kit with the GeneArt® High-Order Genetic Assembly System.
A cloning efficiency of over 90% was observed when assembling 10 fragments of 5 kb each in pYES1L. The fragments were amplified with Platinum® PCR SuperMix High Fidelity and assembled into pYES1L using the GeneArt® High-Order Genetic Assembly System the cloning efficiency is over 90%.
The DNA Oligo Designer is an intuitive, web-based tool to guide you when you are designing your desired DNA molecule. The tool minimizes planning time, identifies potential pitfalls, and performs in silico cloning. The DNA Oligo Designer also provides a graphic representation of the final assembled molecule as well as a downloadable GenBank file.
The DNA Oligo Designer also sends your required DNA oligonucleotides directly to your shopping cart for easy ordering in countries where this functionality is available.
For Research Use Only. Not for use in diagnostic procedures.