Several articles in the synthetic biology section of the IDT DECODED newsletter present methods for cloning double-stranded DNA into plasmid vectors (See the Additional reading sidebar below). The procedure is used for sequencing, building libraries of DNA molecules, gene and non-coding RNA expression, creating synthetic genes and genomes, and many other applications. These articles have reviewed the Gibson Assembly™ method, cohesive-end, and blunt-end cloning techniques. Here we discuss assembly PCR, a method commonly employed for constructing synthetic genes.
Why assembly PCR
Assembly PCR, using synthetically derived DNA, is a very flexible technique for producing novel gene sequences. Single-stranded oligos or a mix of single- and double-stranded DNA are used to produce longer genes of up to several thousand base pairs. The approach can also be beneficial for assembling constructs with modular elements, such as antibodies (see the article, A Next Generation Understanding of Immune Response).
Assembly PCR is also interesting because overlapping sequences can be joined without the need for restriction sites, and one can take advantage of robust PCR reagents and methods. However, because assembly PCR usually involves putting together many short fragments, experiments require careful planning and substantial optimization to be successful. There are several protocols available that use PCR methods and reagents to assemble genes. This article focuses on a two-step approach by Xiong et al. [1–3] (see below, Two-Step Assembly—How It Works). However, most of the considerations are also applicable to other assembly PCR protocols.
Overall flexibility and the low cost of standard oligonucleotides make assembly PCR seem like an easy choice for gene construction. However, there are several considerations that make the technique, in practice, more challenging. In addition to design and logistics factors, the success of assembly PCR is affected by the same factors that affect regular PCR.
Annealing temperatures. Overlapping sequences should have annealing temperatures (Tm) ideally between 60 and 70°C and within 5°C for all termini of the DNA elements being assembled.
Oligonucleotide characteristics. GC content, secondary structure, and repetitive sequences can affect annealing, amplification, and cloning so some sequence optimization may be necessary for successful assembly. In many cases this can be accomplished following existing knowledge and guidelines for PCR. Use the free IDT OligoAnalyzer® Tool, available on the SciTools tab at www.idtdna.com, to quickly analyze the DNA ends for these properties.
PCR conditions. Reaction conditions can be optimized for assembly PCR. Adjusting DNA, dNTP, Mg2+, and enzyme concentrations may be helpful, and inhibitors of PCR, such as chelators and organic solvents, should be avoided.
Primer concentration. Commonly, the outermost primers in an assembly PCR are at higher concentrations, approximately 30 pmol, for amplification of the overall construct, and the internal primers or double-stranded DNA is kept at lower concentrations, approximately 1.5–2 pmol (Figure 1) .
Sequence errors. Another concern with assembly PCR is that a subpopulation of the synthetic oligonucleotides contains small errors that arise during synthesis. The low rate of these errors in quality oligonucleotides is typically not an issue for PCR amplifications because the vast majority of amplified products will be correct. However, for assembly PCR, the statistical probability of one or more of these errors showing up in the final sequence increases with the number of oligos assembled, as well as the lengths of oligos used.
Sequence errors due to oligonucleotide starting materials can be mitigated by using special, high-fidelity oligonucleotides such as IDT Ultramer® Oligonucleotides that are well suited for gene assembly. Including a proofreading DNA polymerase in the PCR will reduce the introduction of additional errors.
A new method, using sequence-verified gBlocks® Gene Fragments, also will reduce sequence errors, and eliminate the need to assemble and clone the smaller fragments in the first assembly step.
Assembly PCR using gBlocks Gene Fragments
A new product from IDT offers a high-fidelity solution that replaces the first step in the two-step assembly PCR approach. gBlocks Gene Fragments are synthetic, linear, double-stranded DNA that are sequence verified using the Sanger method, and are ready to clone using a variety of methods. gBlocks Gene Fragments offer the same sequence flexibility as oligonucleotides, but at a much higher fidelity, and they are available in lengths from 125 to 3000 bp. Thus, the gBlocks fragments can be used to replace the ~500 bp, Step 1 assemblies completely. This provides a substantial savings in time and money spent designing, assembling, and sequencing the Step 1 sequences. The researcher simply orders the desired sequences on the IDT website, and in 2–8 days the gBlocks Gene Fragments are delivered. For a description of how this approach has already been used by one research group, see the research profile article, A next generation understanding of immune response.
Two-step assembly—how It works
Step 1. Single-stranded oligonucleotides, 60–120 nt, designed with short overlapping sequences, are assembled using PCR reagents into approximately 500 bp sequences. The assembled, now double-stranded fragments are then subcloned into a plasmid vector. In fact, for genes <1 kb, this first stage assembly should be sufficient for complete synthesis. However, when directly combining multiple short oligonucleotides into sequences >1 kb, the chances of random errors in the final construct increases.
Step 2. For constructs >1 kb, a second round of assembly is used to join error-free, 500 bp, subfragments that were subcloned and identified by sequencing from the first round of assembly. For this step, the double-stranded elements must also contain overlapping sequences at their termini. The subcloned sequences are isolated from the plasmid either by PCR or restriction digest—PCR using a high fidelity polymerase is typically recommended because it eliminates the need to include restriction sites in the design. The double-stranded subfragments are joined in a second round PCR reaction to create the desired larger sequence, and included primers amplify the construct. This second step of assembly is followed by another round of cloning and sequencing (Figure 1).
Figure 1. Two-step assembly PCR. Step 1: Oligonucleotides with overlapping sequences, approximately 20 bp and with similar Tm, are assembled into double-stranded DNA, 500–1000 bp, using PCR. These fragments are then subcloned and sequenced. Inserts from clones with the correct sequence are isolated by restriction digest or PCR amplification (recommended). Step 2: The double-stranded components are combined, along with amplification primers that will amplify the entire desired construct, and assembled in a second round of PCR. The products are then cloned and sequenced, yielding the final construct.
The main advantages of this approach are that the 500 bp fragments from the first step are easy to clone and sequence, and only subfragments that are 100% accurate are used for assembling into larger gene constructs. The disadvantages to this two-step assembly PCR approach are: 1) large assemblies still involve combining large numbers of oligonucleotides in the first round of assembly, and 2) time-consuming and expensive cloning and sequencing must be performed after both the first and subsequent rounds of assembly to obtain the final construct.