The polymerase chain reaction (PCR) is the backbone of many lab techniques. In short, it allows for the exponential amplification of a specific segment of DNA. Through the use of primers encoding restriction enzyme sites, these amplified fragments are used in downstream cloning procedures, usually leading to the insertion of one, maybe two, PCR fragments into a plasmid. It’s pretty quick and painless, and even accessible by a high school lab volunteer!
But what do you do when you want to construct your own synthetic genes from a variety of DNA fragments? Or if your final product is going to be really large? You certainly don’t want to have to piece everything together via restriction enzyme digest and ligation – that’s incredibly time-consuming and could lead to a slew of mistakes, including incorrect assembly order of the DNA fragments. So what’s a molecular biologist to do?
Polymerase cycling assembly (PCA), or assembly PCR, is PCR’s way cooler older sibling. While both use much of the same technology and reagents, the goal of PCA is to assemble two gene-sized pieces of DNA into one piece for easier cloning.
We all know that PCR cloning involves forward and reverse primers to amplify smaller products and then assembling the fragments via restriction enzyme digestion and ligation. However, for PCA, primers are initially designed so that your oligonucleotide PCR products (about 50 bp) will have an overlapping sequence of 20 bp to the adjacent segment, either upstream or downstream. Then you throw all of these overlapping bits into a ligation reaction. DONE. That means using PCA you can combine many DNA fragments at once to get a final product up to several thousand bp long without relying on restriction sites, as well as synthetic genes or even entire synthetic genomes, minus any scar sequences in the final product!
How Does Polymerase Cycling Assembly Work?
PCA is pretty much like PCR, which is super handy! The main PCA protocol is a Two-Step Assembly, and whether or not you need the second step comes down to the size of your final product. Let me explain…
The goal of Step 1 in the Two-Step Assembly is to piece together sequences up to approximately 1kb. To begin, you must first:
(1) figure out the order of genes you want to stitch together.
(2) design primers (forward and reverse) that will result in a 20bp overlapping sequence with the adjacent piece of DNA.
(3) generate these DNA fragments by PCR (common end size is ~ 500bp).
Once design the short oligonucleotides that overlap with their neighbors, you set up your assembly reaction like a standard PCR, with your template being the mix of overlapping oligonucleotides. Following sequencing to ensure accuracy, these long DNA fragments can be subcloned into a plasmid vector for future downstream use. So if you know your end product is going to be less than 1kb, there’s no need for further action!
However, if you’re planning on assembling a DNA product that is ultimately more than 1kb, you’ll need to pause and set up Step 2. Not to worry! This will be extremely similar to Step 1, with the goal of simply elongating your DNA strand to include all of your genes of interest.
For Step 2, you gather up all of your sequenced <1kb fragments of interest. So if you subcloned them into a plasmid vector after Step 1, go ahead and isolate the fragment using high fidelity polymerase PCR….ready? Good. Now, because you’re an amazing scientist and properly planned out your cloning experiment, all of these fragments should have overlapping termini. In which case, you throw them plus primers for amplification all into a second round of PCR and voila! The final product is cloned and sequenced. BOOM.
If PCA is PCR’s cooler older sibling, the Gibson assembly is their hipster, bad a$$ cousin.
Gibson assembly was developed by Dr. Daniel Gibson in 2009, and allows for the joining of multiple DNA fragments in a single, isothermic reaction. I swear, this has got to be the most awesome way to assemble DNA fragments for cloning. It has saved my butt a thousand times and has never failed me. Seriously. How many techniques can you say that about?
Here’s the low-down: you start with DNA fragments that you want to join together, again with overlapping bp. You then throw in a cocktail mix (you can buy pre–made here) that includes exonuclease, DNA polymerase, and DNA ligase.
As you can probably guess, the exonuclease digests back the 5’ ends, enabling the complementary overhangs to anneal, the DNA polymerase closes any gaps in the strands, and the ligase seals the nicks. Aside from the premade cocktail, do you want to know what the best part of this set up is? The whole reaction incubates at 50oC for 10-60 min (depends on the Gibson Assembly Master Mix manufacturer’s recommendations), the ultimate “set it and forget it”. It’s easier than making brownies from a box.
Anything Else to Be Aware Of?
Not really. If you’re familiar at all with PCR, then those same rules apply for PCA. For instance, similar to PCR primers, PCA oligonucleotides must be able to anneal to complementary fragments of the target sequence, have similar melting temperatures, be hairpin free, and not be too GC rich. Most protocols also suggest that overlapping oligonucleotides have annealing temperatures between 60 and 70oC.
As has been pointed out, PCA involves the assembly of any number of oligonucleotides at a variety of lengths. While amplification errors may not show up in PCR products, the statistical likelihood of sequence errors in PCA products is a point of concern. However, there are thankfully services through companies that make it a business to take care of this stress for the researcher. For instance, IDT offers the generation of gBlocks® Gene Fragments that are 125-3000 bp, so depending on how long you desire your final construct to be, you can either (a) essentially have IDT make your final oligonucleotide product for you (<3kb) or (b) assemble a much larger construct knowing that you are using reliable oligonucleotides to do so (>3kb).
So what do you think? Are polymerase cycling assembly or Gibson assembly techniques that you use often? Comment below!
Xiong et al. (2004) A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res. 32(12): e98. doi: 10.1093/nar/gnh094