How Custom, Double-Stranded DNA is Replacing High Quality Long Oligos
The emerging field of synthetic biology has been rapidly changing the technologies that researchers use for gene assembly and is driving demand for even cheaper and more efficient methods. Most labs that work with DNA on a regular basis have experience creating artificial sequences by assembling single-stranded DNA oligos into double-stranded constructs using PCR-based methods. As oligonucleotides offer good sequence flexibility and are relatively inexpensive, this approach has made sense for decades.
However, PCR assembly is not always straightforward. When working with large assemblies, oligonucleotides must be designed to have comparable annealing temperatures, and must avoid secondary structure in overlapping regions. This means that planning design strategies can take a substantial amount of time. There are also numerous ways that purification and assembly steps can fail, leading to costly material waste and experimental delays.
Oligonucleotide quality is also essential for success, as is the selection of an appropriate assembly method. Sourcing your oligos from the right vendor and using up-to-date, rather than traditional gene assembly protocols, limits disappointment and rework. Choosing a low price oligo may result in a cheaper initial assembly cost, but it is also very likely lead to introduction of sequence errors, requiring more downstream screening as well as sequencing and site-directed mutagenesis costs. In other words, the money saved on cheap oligos will be spent elsewhere on time and other resources to generate an accurate sequence.
If you do decide that assembling oligonucleotides is the correct approach for your project, purchasing high quality oligos will improve assembly success. As an example, Ultramer® Oligonucleotides from IDT, which have the highest coupling efficiency in the industry, will deliver more full-length product than other oligonucleotides (Figure 1), leading to higher assembly success and less downstream screening.
Many protocols recommend using oligos in the 60–80mer range, which is partly because many oligo chemistries are deficient at producing longer sequences. Ultramer Oligonucleotides of 100–120 bases have a much higher sequence fidelity than even 60mer oligos made using standard chemistry. Thus, use of these longer, high quality oligos can reduce the overall number of oligos required for the assembly reaction while also improving assembly success. They can also significantly facilitate the construction of more complex structures. However, a coupling efficiency that is a remarkable 99.5% still translates to only ~60% full-length product for a 100-base oligonucleotide (Figure 1). This means the number of correctly assembled constructs can vary from 30% to 80%, depending on sequence and size. In addition, these high quality oligonucleotides come at a price per base that can make assembly of large constructs costly. Researchers have been working within these limitations for decades. However, there was obvious room for a better gene assembly solution.
Figure 1. gBlocks® Gene Fragments Provide More Full-Length Product. The graph shows the effect of coupling efficiency on yield with increasing oligo length. Ultramer® Oligonucleotides provide the highest yield of full-length sequence for gene assembly. Gene assembly applications typically use oligos from 50 to 120 nucleotides long (gray shaded region), and higher quality Ultramer Oligonucleotides will ultimately generate fewer sequence errors in these applications. However, double-stranded gBlocks Gene Fragments use a different manufacturing process that results in 100% full-length product. In addition, when cloning a gBlocks Gene Fragment, on average, 80% of the clones produced (90% for fragments <500 bp) will have 100% correct wild-type sequence, regardless of the length of the cloned fragment, providing a compelling alternative to single-stranded DNA for construct assembly.
Custom dsDNA Fragments
If your lab has been using oligonucleotides or PCR for gene construction and cloning, you may want to consider a new class of custom dsDNA fragments that is now available. Developed about 2 years ago by IDT, gBlocks® Gene Fragments offer a streamlined way to assemble synthetic genes with greater ease and reliability, as compared to oligonucleotides joined by assembly PCR. gBlocks Gene Fragments are also compatible with most gene construction methods, so you will not have to make major changes to your construct design and assembly protocols to see how they can improve experimental results.
gBlocks Gene Fragments are available up to 2 kb in length and are easy to order. Because they are double-stranded fragments, they can be used exactly as you would use a PCR product; they can be assembled into larger constructs, cloned by conventional methods, or used as delivered for applications requiring linear dsDNA. Because the sequence you specify is synthesized using a unique manufacturing method, gBlocks Gene Fragments are not limited in quality or length by coupling efficiency in the same way as oligonucleotides—100% of the product is full-length (Figure1). They ship in a few days as a sequence-verified product in which, on average, 80% of the DNA population provided is the requested sequence regardless of length.
Using longer, custom dsDNA as a starting material allows you to turn otherwise complex assemblies with many parts into simpler reactions, or to order entire target sequences as complete gBlocks Gene Fragments ready for cloning or other uses. This is a major improvement over the assembly of many oligos and can eliminate the frustration of troubleshooting failed oligo assembly reactions.
Experimental Applications Made Easier
Being able to order dsDNA that has high fidelity and is sequence verified allows researchers to get to the downstream applications that matter to them more efficiently. gBlocks Gene Fragments have been used in a wide array of demanding applications, including their prominent use in CRISPR/Cas 9 targeting and genome editing. In many of these examples, gBlocks Gene Fragments have been used as CRISPR guide RNA (gRNA) expression cassettes, where their size and sequence flexibility makes it easy to generate the gRNA under control of a U6 promoter for insertion into an expression vector, or downstream of a T7 promoter for expression in E. coli or by in vitro transcription. gBlocks Gene Fragments also allow for easy codon optimization and assembly of the bacteria derived Cas9 for use in other organisms.
Researchers who have used gBlocks Gene Fragments have commented on the significant differences between these dsDNA fragments and other technologies. Dr Mark Rieder, VP of Operations at Adaptive Biotechnologies, called this new tool “a game changer.” Adaptive Biotechnologies uses gBlocks Gene Fragments to optimize their next generation sequencing assays, and Dr Rieder told us that they knew this technology would allow them to make a better assay and do so faster than if they made the necessary constructs in-house. In addition, the sequence verification that IDT performs on gBlocks Gene Fragments meant that Adaptive Biotechnologies’ scientists did not have to worry about assembly errors.
These are only 2 examples of gBlocks Gene Fragments applications. Other interesting applications of these dsDNA fragments are listed in Table 1 and several have been highlighted in the IDT DECODED newsletter. We also provide a long list of citations and downloadable protocols at www.idtdna.com/gblocks.
More Innovations to Come
On a final note, the flexibility of gBlocks Gene Fragments continues to expand. An excellent example of this is the recent availability of gBlocks Gene Fragments that include degenerate bases—up to 18 consecutive N or K mixed bases, a feature that was largely driven by customer requests. These DNA fragment libraries are used by academic and commercial laboratories to, for example, test the function of variants in a nucleotide or amino acid sequence. gBlocks Gene Fragment Libraries allow these scientists to assemble large numbers of constructs for pennies or less each. What do you want to do with gBlocks Gene Fragments? We would like to know; just email us at email@example.com.
CRISPR genome editing
Yang L, Yang JL, et al. (2014) CRISPR/Cas9-directed genome editing of cultured cells. Curr Protoc Mol Biol, 107(31):1–17.
Dickinson DJ, Ward JD, et al. (2013) Engineering theCaenorhabditis elegansgenome using Cas9-triggered homologous recombination. Nat Methods10(10):1028–1034.
Shlyueva D, Stelzer C, et al. (2014) Hormone-responsive enhancer-activity maps reveal predictive motifs, indirect repression, and targeting of closed chromatin. Molecular Cell, 54(1):180–192.
NGS assay development
Carlson CS, Emerson RO, et al. (2013) Using synthetic templates to design an unbiased multiplex PCR assay. Nat Commun, 4(2680).
Huang H, Suslov NB, et al. (2014) A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore. Nat Chem Biol, 10(8):686-691.
High resolution melt
Bruzzone CM, Tawadros PS, et al. (2013) Enhanced primer selection and synthetic amplicon templates optimize high-resolution melting analysis of single-nucleotide polymorphisms in a large population. Genet Test Mol Biomarkers, 17(9):675–80.
Table 1: Published gBlocks® Gene Fragments Applications. This list is by no means exhaustive. We apologize if your application escaped our scrutiny, but we’d like to hear about it! Please tell us about your original use of gBlocks Gene Fragments (firstname.lastname@example.org) and we will share it with the community.
Author: Hans Packer, PhD, is a Scientific Writer at IDT.