Triple Labeled Oligos-Put Your Fluorescence in Perspective!

Allele is now distributing to its customers the newest technology in RT-PCR published less than two weeks ago. It is a RT PCR probe that utilizes fluorescence resonance energy transfer (FRET) and TaqMan methods in order to provide an internal positive control (IPC) for real-time PCR assays. This new RT-PCR method consists of forward and reverse primers, a regular TaqMan probe, and the new triple-labeled FRET-TaqMan Probe Probe affixed to a plasmid, which negates the uncertainty in the presence and effects of amplification inhibitors that could generate a false negative in your experiments. Two emission sources are all that are required for the FAM and Cy5.5 fluorescence signals at 530 nm and 705 nm respectively. You do not need color compensation software, multiple excitation sources, or even a high tech laser to initiate excitation with this system.

If your RT PCR instrument only has one light source to excite the fluorophore then the FRET-TaqMan method is the only way to exploit the IPC technology. Fortunately Allele Biotech is selling these triple-labeled probes at a great price! Put your fluorescence in perspective and know your true RT-PCR results with these Specialty Oligos from Allele Biotech!

Tags: fluorescence signal, FRET Probe, FRET TaqMan Probe, internal positive control, IPC, led excitation, multiplex real-time PCR, probe, real-time PCR, RT-PCR, special oligonucleotides, TaqMan Probe, tripple-labeled oligos

Four ways to clone your PCR products, which one should you use?

Cloning of a PCR fragment is typically achieved by one of the following methods:

1) TOPO cloning using a TOPO blunt vector for PCR products generated with Pfu or many other high fidelity thermostable DNA polymerases, or TOPO TA vector for PCR made with Taq. The efficiency is high and so is the cost partially due to the exclusivity of Life Technologies (Invitrogen) to offer topoisomerase-based cloning vectors. TOPO kits always include competent cells, controls, etc. with the topoisomerase-conjugated vector, further increasing the prices to about $25/reaction. The background of TOPO cloning is normally low as the enzyme-linked ends of the linear vector DNA do not allow self-ligation; however, occasionally circular plasmid formation proceeds through recombining one end of the linear vector to a vector sequence hundreds of bases downstream of the other free end.

2) Restriction digesting PCR fragment for ligating to cloning vector. This method allows you to have the freedom to choose virtually any plasmid vector that immediately suites your experiments intended for using the clone. Unfortunately the efficiency of cutting restriction sites introduced to the ends of linear PCR DNA pieces is often extremely low, even with extra bases added to the outside of the restriction enzyme sites. Background ligation by vector self-ligation or erroneous ligation to contaminated DNA becomes prominent when properly cut insert DNA is not present in sufficient concentration due to poor restriction digestion. The cost should be the lowest using this method, given that you don’t have to do repeated digestion with increasing amount of restriction enzymes and ligation with batch after batch of ligase.

3) End fusion via recombination assisted by end-chewing DNA polymerase or other DNA enzymes. The most actively promoted commercial product for this method is the In-Fusion line from Clontech (Takara). The cost of using the In-Fusion kits is somewhat lower than the TOPO kits, still above $12/reaction in most cases even without accompanying competent cells. At least there is an option of just buying the kit without competent cells, which is basically some dried-down viral DNA polymerase in separate tubes. Because in theory enzyme-assisted recombination can occur between any homologous sequences at the ends of linear DNA, the user has freedom to choose any vector. In practice however, it is questionable how much the added DNA polymerase actually help the efficiency since in our hands we found sometime the no-enzyme controls worked better than using the supplied enzyme. Furthermore, we also observed vector dependence of cloning efficiency.

Preannouncement: Allele Biotech will soon offer a linear form of DNA for both entry point PCR cloning (i.e. putting linear PCR product into a circular vector so that subsequent restriction digestion at the ends of the PCR fragment will be easy) and basic bacterial protein expression. There will be no enzyme required at all and the cost will be much lower than similar products from other suppliers.

4) TA cloning is a simple method of cloning DNA fragments created by a PCR reaction catalyzed by the Taq DNA polymerase. The PCR product with 3’ overhanging A base is ligated to a linear vector with overhanging 5’ T. This method was devised after the discovery that the 3’ ends of PCR products generated by Taq polymerase contain an unmatched A base added by the terminal transferase activity of Taq polymerase. The T/A matching so provided helps increase the ligation efficiency over blunt end ligation such as ligation between Pfu-generated PCR and vectors cut by EcoR V or Sma I. Therefore, even when high fidelity enzymes are used for PCR, many researchers choose to add the extra A base by incubating the PCR product in the presence of Taq for about 15 min. Even though a linear vector with a 5’ overhanging T is not trivial to produce, the cost of TA cloning vectors is still much lower than TOPO or end-fusing vectors. Allele Biotech’s just-launched TA cloning vector also utilizes a LacZ-based blue/white selection. The background ligation rate is very low while ligation with insert typically results in a few hundred colonies by standard procedures using Allele’s Extreme Efficiency competent cells.

Tags: end fusion, LacZ-based blue/white selection, restriction digesting, TA cloning kit, Taq DNA polymerase, TOPO cloning

Launch of Allele’s Newest Product Group: Special Chemicals

It has only 4 products now, will be a dozen soon, then a few hundreds to thousands in a few months if all goes well. If not, just the oligo chemicals available now should already provide tremendous value to oligo producers because iPCR, Real-time PCR are getting more and more used, requiring large number of probes with these modifications we products can do, but at previously unthinkable low prices. Read about it here.

Tags: amino group, biotin, blocking elongation, capturing oligos, Dabcyl, Dabsyl, FAM, hybridization, iPCR, matching sequences, oligos, probes, real-time PCR

Swine Flu

Today the World Health Organization declared that the Swine Flu-(Influenza A(H1N1)- pandemic has reached 21 countries. The disease that is thought to have originated in Mexico has claimed 29 lives in the infection of 822 people in that country alone.

As San Diegans and as global citizens, Allele Biotech wishes to covey its sympathy to all people affected either directly or indirectly by this pandemic. As a company Allele Biotech is doing its part to help eradicate the existence of such an ominous virus that threatens people’s health and livelihoods. Recently Allele Biotech negotiated a deal with a distributor in Baja. The agreement that generated Allele Biotech De Mexico could not have come at a more fortuitous time. Our Baja researchers are feverishly working on this worldwide effort to elucidate, control, and prevent the A H1N1 that is currently affecting all of us.

Allele Biotech De Mexico is working in conjunction with Allele Biotech of San Diego to fight Swine Flu. On Friday, May 1, 2009 several researchers and technicians worked into the night on custom designed oligonucleotides that are being used right now to research this virus. We are thoroughly optimistic that this virus will soon be under control and we are honored we could help in this urgent endeavor.

Tags: A H1N1, A H1N1 pandemic, Allele Biotech researches swine flu, pandemic, Swine Flu Mexico

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.

Tags: dsDNA, fast turnaround, insert, oligo, PAGE, PCR, primer, probe, siRNA, vector