oligo
Competition from the Marketplace to the Courtroom
The hottest subject in the biological research equipment field has to be whole genome sequencing; hence it is no surprise that companies execute mergers and acquisitions in order to position themselves to go after their competitors in an attempt to corner this valuable market.
A bit of the background history: Illumina was started a decade ago to build DNA chip arrays by people with experience at Affymetrix, when the latter was the first and absolute leader in the DNA chip field. For years, rather than providing DNA chips, Illumina was known for generating revenue by selling oligonucleotides at 20% of the prevailing market price, essentially starting the low end oligo market. Just three or four years ago, it was a front page promotion on Invitrogen’s website to sell Illumina’s oligos through a production/shipping alliance, a cooperation previously unheard of in our field for such low price, non-commodity products. This move quite probably contributed to the decisions made by the more dedicated oligo company, IDT, to acquire local oligo production houses and move to the West coast (Allele opted out of such an acquisition and later did one of its own by taking over Orbigen and since moved into the viral systems and antibody fields). At that point when whole genome sequencing technologies were becoming mature and marketable, Illumina had performed brilliantly in out competing the previously dominant chip supplier Affymetrix, acquired Solexa, and quickly moved into the whole genome sequencing with Genome Analyzer and Genome Analyzer II, a move Affi’s management probably regretted not making.
In the years roughly around 2005-2007, Applied Biosystems, Inc. (ABI) was developing its own genome analysis equipment, the SOLiD system. It surely had a solid base to build on from its strong leadership in providing sequencer and analyzers for many years. Earlier in the year Invitrogen and ABI merged to form Life Technologies, pitching Invitrogen (now LifeTech) and Illumina in a collision course in battle for dominance in genomic analysis. In September, LifeTech brought suit against Illumina for patent infringement; in October Illumina countered with suits of its own. While the fight in court may be long and only sprinkled with occasional fireworks, the competition in the market could be fierce and should ultimately decide on whose technology is superior and offered at better prices. From the technical presentation made by sales teams to us during on site seminars, Solexa’s science sounded better. I was sitting next to Jay Flatley, CEO of Illumina at a San Diego biotech CEO dinner, and heard him predicting that the technology would advance and in a few years, one could get their own genome sequenced for about a thousand dollars, ~10% of the current cost! That’s simply innovation and competition at work. But watch out, a new wave of sequencing technologies based on single molecule capture might make the Illumina and LifeTech courtroom argument a moot point in the market.
HPLC Purified siRNA with Known RNAi Effects at $149/12.5nmol
RNA oligo is significantly more difficult to synthesize than DNA oligos, mainly because the efficiency of coupling each new ribonucleotide during RNA synthesis is a few fold lower than deoxyribonucleotide during DNA synthesis. Typically, there is an ~10% chance a DNA oligo of 21 bases will have a mutation (most frequently a deletion mutation); for an RNA oligo of 21 bases, as in an siRNA pair, such chance is much higher. Furthermore, after combining the sense and antisense siRNA strands, some RNA molecules will remain as single-stranded thereby not fitting for the RNAi apparatus.
RNA interference is a dose-sensitive process — specificity of gene silencing is meaningful only relative to the active concentration of siRNA used. When the concentration is too low, even the most effective siRNAs would fail to cause gene expression knockdown; when too high, non-specific effects will be duly observed. Therefore, it is essential that the concentrations of siRNAs are measured correctly. When doing so, one must consider not only what the apparent concentrations are by OD260 reading, but also whether the RNA strands are of full-length and whether only dsRNA molecules are counted. This issue might not affect data interpretation if appropriate controls are included in one set of RNAi experiments, but it could have significant influence on conclusions if data from different experiment sets or labs are compared or combined.
HPLC purification currently provides the best means to remove RNA molecules with deletions or remain single-stranded, however, the price tag added by most reagent providers for such treatment has been prohibiting because manufacturers either need to start synthesis at a much bigger scale to obtain promised amount, or they do not promise the delivery quantity at all. The phosphoramidites (oligo building blocks) for RNA synthesis can be 10 times or more expensive than for DNA. Some companies offer alternative purification methods such as a cartridge type device, but they can only remove salt and small impurities, not RNA oligos of shorter lengths accumulated at each cycle of amide coupling. The AllHPLC siRNAs within Allele’s RNAi product line, pre-validated or custom made, are uniformly HPLC purified with 5 OD or 12.5 nmol of double-stranded, annealed siRNA delivered. Allele passes to customers the cost savings from manufacturing our own RNA amidites and other reagents for oligo synthesis. The pre-validated HPLC purified double-stranded siRNA is offered today at $149/12.5 nmol.
Before purchasing siRNAs, even at a low cost of $29 per pair of HPLC purified control siRNA from Allele, researchers still need to consider how well their cells can be transfected. For hard-to-transfect cells, lentiviral vectors carrying a shRNA expressing cassette is often a better choice. To establish stable cell lines, plasmid vectors should be considered. For low cost target screening, the PCR format linear siRNA expression cassettes have advantages.
Economy and Your Research: Carpets and Oligos
Do you believe in six-degrees of separation? If you really don’t care how close you are related to Roger Tsien or Bill Gates or the dean of your graduate school, maybe you are still curious about how the economy downturn, oil production, and floor carpet production got to do with you–not just in the sense how the job market is shaping up, but also how your lab research budget and how your DNA oligos are served.
To illustrate how events far and away can influence your daily activities, just use oligos as an example. Starting in 2008 when the oil price was still near its peak (remember paying $4+/gallon?), it became too expensive for carpet producers to continue using petroleum for manufacturing carpets. They switched to some other source or halted business altogether. Side effect was production and supply of Acetonitrile (ACN) dried up. Yes, one of the most commonly used organic solvent is a by-product from making carpets. That, combined with facility shutdowns in Northern China in preparation of the summer Olympics (for clean air) and in Florida by a major hurricane, the price of 4 liters of Acetonitrile changed from ~$40 to about $400 plus lots of begging. This event alone pushed individual customer based (as compared to large scale or prefabricated) oligo businesses like Allele’s to be at a loss.
Eventually the situation changed, price went back to about $90/4L, but not before a long period when Acetonitrile was completely unavailable and alternative solvent had to be used. Long story short, that was some storm to whether! If you didn’t feel it in price or service from Allele Oligo, good, that means we did a fair job shielding the wind and shouldering the pressure from the collapsing roof.
Everything really is connected, sometimes by a few degrees less than you would imagine.
Allele’s mottos: care about the environment, help everybody whenever we can, do the right thing even when nobody is looking, have fun, and contribute to the good of mankind through science and innovation.
Something you should know about oligos
Oligos are made from 3’ end to 5’ end by nucleotide-wise coupling. Each coupling cycle involves about half a dozen moisture-sensitive chemicals and takes about 15 minutes to complete when 96 oligos are being synthesized at the same time on one machine. Like most chemical reactions, couplings do not reach 100% efficiency; in consequence, about 1% of the oligos would have an unsuccessful coupling at any given position and therefore missing that base. There are “capping” steps designed to prevent oligos having an unsuccessful coupling from continuing to elongate; but in practice, capping can only reduce incomplete oligos in the final pool, not eliminate them.
In PCR reactions, primers with mistakes typically have less chance of pairing with template than those with perfect match. Increasing annealing temperature may prevent primers with deletions from participating in PCR reactions. However, oligos that miss 5’ end restriction site but have no internal deletions will not be selected against by higher annealing temperature since initial annealing is not affected. Purification of oligos by PAGE can effectively remove oligos with deletions in any position, albeit not eliminate them. For cloning purposes, purifying oligos typically makes the post-ligation steps (i.e. inoculation, minipreps, and sequencing) much easier.
At Allele Biotech Oligo Services, we use top quality chemicals from Glen Research and extensive coupling and washing steps in order to synthesize oligos with as few mistakes as chemically possible. Most oligo mistakes occur in individual molecules, which you may encounter by chance. Sequencing a few more colonies for a cloning project is the easiest and fastest way to achieve desired results if a mistake is found in the first round of sequencing. If the customer prefers remaking oligo, we honor our 100% guarantee policy with replacement of all our oligos shorter than 45 bases and the longer ones with purification. We always appreciate our customers’ feedback.
General suggestions for using oligo primers for cloning
* Use more stringent PCR annealing conditions if possible.
* Purify oligos, especially long primers.
* Even though higher cost on oligos, much lower costs and less time on minipreps and sequencing.
* Since mutations happen randomly, sequence a few more colonies could result in identifying desired plasmid
Sometimes it is very difficult to clone PCR products by restriction digestion and ligation. Restriction enzymes do not cut well near the end of linear DNA even with extra bases added 5’ to the restrictions sites. It is almost always helpful to clone the PCR fragment into a PCR cloning first. Cut with designed restriction enzymes and send only those showing insert of correct sizes for sequencing.
Oligo mutations augmented by E. coli during colony selection. The following was a real case using Allele oligos to create genes encoding fusion antibody chains. The strategy was to fuse a humanized antibody heavy chain to a light chain by a pair of oligos that would overlap cDNAs for both chains. The vector was pBluescript II, where insert could disrupt an expressed beta-galatosidase, thus changing the color from blue to white in the presence of IPTG and X-gal. After minipreping and sequencing dozens of colonies, all plasmids had frame-changing mutations in the junction region, which were seemingly introduced by the primers. Worrying about the oligo quality, we checked oligo synthesis records, including reagent log of that run, Trityl color indication record (indicator of coupling efficiency of up until the last base), gel pictures of oligos made in the same batch, feedback record from other customers using oligos from that day. We could not find anything unusual from the records. We decided to remake the oligos. After two weeks of work to regenerate plasmids for sequencing, the same results were obtained—all plasmids had mutations in the same region. There seemed to be nothing else we could do but to remake the oligos with somewhat different designs, e.g. shifting the overlapping regions slightly, or shortening the oligos a little bit, and performed PAGE purification this time. The results were the same once again.Then it occurred to us that maybe the expression of the protein from the pBluescript vector caused toxicity to E. coli and therefore forced the bacterial cells to either select those clones with frame-shifting mutations or create mutations by themselves during growth. Without the option of changing the vector choice, we simply used a different competent cell strain that does not support expression from the promoter on pBluescript. We did it with oligos from different preps, purified and unpurified, in an attempt to obtain as much information as possible about oligo use for our customers and our own future research.The result, all plasmids sequenced were completely correct.
Other Cloning Example Cases:
Case 1:
Aim: To synthesize a 1,650 bp gene from oligos.
Design: Design 36 overlapping oligos of 60 to 80 bases long.
Experiments: Difficult to do PCR in one piece with all oligos. Switched to 3 separate PCR for about 550 bp each.
Results: Sequenced plasmids from colonies with each of the 3 parts cloned into PCR cloning vector: Part I: 2 plasmids sequenced, both with mutations; 1 more sequenced, correct. Part II: 1 plasmid sequenced, wrong; 1 more sequenced, correct. Part III: 1 plasmid sequenced, with mutation; another sequenced, wrong; 3 more sequenced, 2 correct.
Conclusions: Mistakes coming from oligos are random, there is no prediction exactly how many colonies should be sequenced, but normally sequencing 3 in a group is a good practice.
Case 2:
Aim: To synthesize a 300 bp gene from oligos.
Design: Design 16 overlapping oligos of 60 to 80 bases long.
Experiments: PCR in one piece with all oligos.
Results: Sequenced plasmids from colonies cloned into PCR cloning vector: 2 plasmids sequenced, both with deletions; 3 more sequenced, 1 with deletion, 2 with base change; 2 more sequenced, both completely correct.
Conclusions: When luck is not on your side, sometimes a short DNA still requires a good number of colonies to be sequenced.
Case 3:
Aim: To synthesize a hypothetical gene of 1,800bp from oligo overlapping assembly.
Design: 34 oligos of about 55 bases each direction.
Experiments: Difficult to do PCR in one piece with all oligos. Switched to 3 separate PCR for about 550 bp each.
Results: Sequenced 2 colonies, 1 was perfect from one end for about 900 bases, but a number of mutations found when sequenced from the other end. The 2nd plasmid was 100% correct from the first to the last base over the entire 1.8kb region!.
Conclusions: When luck is on your side, you may hit the jack pot blind-folded.
Case 4:
Aim: PCR-clone 3 human cDNAs into a baculovirus expression vector.
Design: PCR with primers that would introduce restriction sites Not I and Xho I.
Experiments: PCR with standard procedure with high fidelity polymerase, PCR products were then run on gel and the desired bands purified by Allele DNA purification kits.
Results: Two of the 3 constructs were all correct in all plasmids sequenced. Plasmids for the other construct all showed PCR products missing half of the Not I site. Sequencing additional plasmids gave the same results. We then gel purified the primer, repeat the process, and all plasmids sequenced were correct.
Have any insights or comments of your own about using oligos? Let’s share them. Thread away.
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