The art of blowing up protein

The other major issue is the limit of a multiplex PCR. Kapa multiplex PCR website talks 4-plex, 8-plex and 12-plex, but it mentions in the FAQ that 30 is highest mentioned. High-plex 30 amplicons, ≤1000 bp. 30-plex is no way close to 4300-plex. However, this does not mean it is impossible.

Multiplex PCR is often used in conjunction with detection methods (TaqMan etc.), and not for subsequent cloning. So some issues may not apply. Two issues however do apply, artefacts and biases.

On chip multiplex PCR. Parenthetically one paper makes a 100-plex PCR on chip to boost sensitivity (PMID: 21909519). This is a related system and a very plausible approach, however, it would require in situ DNA synthesis capabilities.

Primer dimers.  Primer dimers happen in normal PCR. The way to avoid it is using a higher annealing temperature (longer primers) and to avoid repetitive sequences. The oddity is that the Kapa manual gives an example annealing step as 60 °C. If the PCR were a two-step reaction, the reaction would be more efficient with less chance of noise. That means that AT-rich organisms, like P. ubique, are off. DMSO and betaine allow better PCR specificity especially with GC-rich sequences, so it might be good to go even more overkill with annealing length.

3’ trail primer binding.  Here a new issue arises: the first copy of a sequence might have a primer binding site on its 3’ end for the next gene. This truncated gene would not amplify with the other primer, but would act as an inhibitor soaking up the first primer. Amplicons can act as megaprimers, so it might be as bad. Nevertheless, it is worth worrying about. Furthermore, Taq polymerase does not have strand displacement activity but 5’ exonucleolytic activity. One way to overcome this is to have 5’ protected primers (IDT options), which may interfere with the plasmid construction set and may be problematic for overlapping genes. I would shy away from strand displacing enzymes are they may be non-optimal.

The amplification step. PCR amplification efficiencies differ between primers. I could model what may happen (simulate, BRENDA has all the constants I need). However, PCR reactions with low primer concentration might avoid sequences lagging behind. This combined with the previous problem raises the suspicion linear amplification may be best.

Linear amplification. One thing to avoid is exponentially amplifying biases. One option is to do a single primer linear amplification. At first I would say no as it is really wasteful in terms of primers (which are limited) and the yield would not be enough. Plus it would need a second step in the opposite direction to produce correctly sized sequences. Plus the major issue with high-plex reactions is the low yield of product, so this would be four-thousand fold worse when linearly amplified. However, what is not being considered is the fact that a 4300plex PCR isn’t amplifying exponentially. In 25 µl reaction with 100 ng template and with 200 nM primer pool, there are about 2.5 pM (for 4.6Mbp) genome strands and 50 pM of each primer (of 4300), which only allows for a twenty fold amplification.

How does one go about testing it? There will be smears if it is a nonspecific mess and if it worked. For the final product, NGS may be an option to see bias amplification, but not for routine troubleshooting. Transforming specific rescue strains is too laborious. The best option is RT-PCR of some genes (spread of sizes).

The next step once a pool has been made is to get them in plasmid form.

RE cloning, blunt. Messy products, low yield. Non-directional. Not worth it.

RE cloning, added 5’ site. It would require subsets to avoid cutting the amplicon. Which is would be a pain. Also it means adding to the primer.

Gibson assembly. It requires adding to the primer. This means longer primers, which is okay-ish, but I have an unscientific hunch it may mess up the multiplex primer.

TOPO clone. Directional TOPO is a good option, but has a drawback, there is no good plasmid. Also I am ignoring the 5’ primer protection possibility. The pET is not good as it requires T7 (cotransformation of pTARA plasmid and rescue screen has not been done). The pBAD and the pET have loads of crap at the end. The Aska collection is 10% non-functional due to the GFP tag. The thioredoxin and the hexahistidine tags may be cool, but they are in the way of a clean experiment. Adding a stuff to the primers to override the crap is a no-go due to the 5’ tag in multiplex concern and is seriously inelegant for downstream processes. So the TOPO ligation into pENTR plasmid followed by Gateway into a custom plasmid. It would allow a brief amplification step. A custom TOPO would be ideal, but it costs more than the GeneStrings step itself. A good homebrew TOPO-plasmid has never been achieved. So, disappointingly, a two-step TOPO is the TOPO way (I’ll keep an eye out for new TOPO kits).

P. ubique is not an option as it is too AT-rich and its genes cannot be expressed even with pRARE. Even though this whole idea arose to tackle the pathway holes issue in P. ubique.
I want to try it for T. maritima to find if MetC is the sole racemase, but as there is the structural genomics consortium’s library, it makes it seem a tad redundant.

If I were to do this independently of my project, I would be easy to find a good candidate that:

For now, I’ll call it ORFome library by multiplex linear amplification.  I have been calling it Aska-like library. Although I’d like a better one. Yes, a name is a semantic handle, but there is nothing worse than a bad name. Delitto Perfetto is a cool method name, CAGE isn’t and I rather keep away from backronyms. Portmanteaux are where it’s at, especially for European grants. Geneplex is already taken, but -plex means -fold anyway. Genomeplex? Mass ORF conjuration spell? I would love to coin a name, but I should withhold for taken the fun from later on.