What seems to be going on with RNAi related patents in the US

Reciting Table 1 from Ref 1 and Table 3 from Ref 2:

By the end Nov 2008 it appears that Allele’s patent (US 7,294,504 and 7,422,896) are the only currently granted DNA based RNAi patents. The focus of Allele’s technology is siRNA of 21-23, either in separate sense and antisense strands, or shRNA or miRNA format, thus not covered by the Fire patent or the Kreutzer-Limmer patent. Since these RNAi inducers are not synthesized by chemical reactions, or produced with enzymes or cell lysate in vitro, they do not relate to Tuschl I or II patent groups. Allele Biotech can not guarantee that its interpretation is correct or final by any means; commercial user of any of the related technologies should perform own due diligence.

[1] Charlie Schmidt. March 2007 “Negotiating the RNAi patent thicket” Nature Biotechnology 25 (3): 273-275

[2] Dirk Haussecker. May 2008 “The Business of RNAi Therapeutics” Human Gene Therapy 19: 451-462

Have an opinion? Feel free to share it here.

Tags: gene silencing, H1, lentiviral vector, miRNA, PCR cassette, plasmid vector, pol III, retroviral vector, RNAi, shRNA, siRNA, U6

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

Discovery and Development of GFP Rewarded by 2008 Nobel Prize in Chemistry!

Press Release 8 October 2008

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2008 jointly to Osamu Shimomura, Marine Biological Laboratory (MBL), Woods Hole, MA, USA and Boston University Medical School, MA, USA, Martin Chalfie, Columbia University, New York, NY, USA and Roger Y. Tsien, University of California, San Diego, La Jolla, CA, USA “for the discovery and development of the green fluorescent protein, GFP”.

Glowing proteins – a guiding star for biochemistry

The remarkable brightly glowing green fluorescent protein, GFP, was first observed in the beautiful jellyfish, Aequorea victoria in 1962. Since then, this protein has become one of the most important tools used in contemporary bioscience. With the aid of GFP, researchers have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or how cancer cells spread.

Tens of thousands of different proteins reside in a living organism, controlling important chemical processes in minute detail. If this protein machinery malfunctions, illness and disease often follow. That is why it has been imperative for bioscience to map the role of different proteins in the body.

This year’s Nobel Prize in Chemistry rewards the initial discovery of GFP and a series of important developments which have led to its use as a tagging tool in bioscience. By using DNA technology, researchers can now connect GFP to other interesting, but otherwise invisible, proteins. This glowing marker allows them to watch the movements, positions and interactions of the tagged proteins.

Researchers can also follow the fate of various cells with the help of GFP: nerve cell damage during Alzheimer’s disease or how insulin-producing beta cells are created in the pancreas of a growing embryo. In one spectacular experiment, researchers succeeded in tagging different nerve cells in the brain of a mouse with a kaleidoscope of colours.

The story behind the discovery of GFP is one with the three Nobel Prize Laureates in the leading roles:

Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria, which drifts with the currents off the west coast of North America. He discovered that this protein glowed bright green under ultraviolet light.
Martin Chalfie demonstrated the value of GFP as a luminous genetic tag for various biological phenomena. In one of his first experiments, he coloured six individual cells in the transparent roundworm Caenorhabditis elegans with the aid of GFP.
Roger Y. Tsien contributed to our general understanding of how GFP fluoresces. He also extended the colour palette beyond green allowing researchers to give various proteins and cells different colours. This enables scientists to follow several different biological processes at the same time.

Read more about this year’s prize

Information for the Public
Scientific Background
In order to read the text you need Acrobat Reader.
Links and Further Reading

Osamu Shimomura, Japanese citizen. Born 1928 in Kyoto, Japan. Ph.D. in organic chemistry 1960 from Nagoya University, Japan. Professor emeritus at Marine Biological Laboratory (MBL), Woods Hole, MA, USA and Boston University Medical School, MA, USA.
www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html
Martin Chalfie, US citizen. Born 1947, grew up in Chicago, IL, USA. Ph.D. in neurobiology 1977 from Harvard University. William R. Kenan, Jr. Professor of Biological Sciences at Columbia University, New York, NY, USA, since 1982.
www.columbia.edu/cu/biology/faculty/chalfie/Chalfie_home/
Roger Y. Tsien, US citizen. Born 1952 in New York, NY, USA. Ph.D. in physiology 1977 from Cambridge University, UK. Professor at University of California, San Diego, La Jolla, CA, USA, since 1989.
www.tsienlab.ucsd.edu
Our congratulations to all of them! We are particularly happy for Roger Tsien, graduate adviser of our own Nathan Shaner and a UCSD colleague and teacher of many of us here at Allele.

Getting the most from fluorescent proteins

Fluorescent proteins (FPs) are an indispensable component of the biology toolbox, providing a robust and straightforward method to optically label nearly any protein of interest.

While most FPs can be used for a wide variety of experimental setups and conditions, getting the best quality data from your hard efforts requires some forethought. Here are a few tips to get the most out of FP imaging:

1.Reduce pre-measurement photobleaching.

All FPs photobleach upon exposure to excitation light. Some, like commonly used YFPs, bleach rather quickly, while others, such as Allele’s mTFP1, are substantially more photostable. However, even the most photostable FPs can be susceptible to excessive pre-measurement bleaching if precautions are not taken.

While searching for your favorite cell on the microscope, try to use the lowest possible excitation light intensity. Close the shutter when you’re setting up software or other experimental apparatus, and use short exposure times whenever possible during focusing.

2.Consider pH and other variables.

Most FPs are somewhat sensitive to acidic pH. Some, such as mTFP1 and many of the red FPs, are reasonably resistant to pH changes, while others, such as EYFP, are highly sensitive. If you’re imaging acidic compartments such as lysosomes or plant vacuoles, you’re unlikely to see any fluorescent signal if you’re using EYFP or any other pH-sensitive FP, so choose wisely!

Information on the fluorescence pKa (the pH at which 50% of the fluorescence emission is quenched) of new fluorescent proteins is generally easy to find, so do your homework!

3.Be careful with fusions and linkers.

One big advantage of using FPs is that they may be genetically fused to virtually any protein of interest. While FPs usually have a negligible effect on the properties of their fusion partners, it’s always a good idea to double-check and validate data on new proteins.

If you don’t know where your protein should localize, check both N- and C-terminal FP fusions to be sure they give the same results. If not, validate your localization by other methods, such as antibody staining. If you can devise a functional assay for your FP-labeled protein, this is also a good way to be sure the fusion isn’t causing trouble.

If you’re having problems with a particular FP fusion, try a few different linkers between the FP and the protein of interest. Floppy linkers, such as poly-(Gly-Gly-Ser-Gly-Gly-Thr) frequently work well, but occasionally rigid linkers (such as poly-proline) or other sequences will give better results. Unfortunately, the process of optimizing a fusion construct is largely empirical.If you can put in the effort early in your experiments to produce the best possible FP fusion, you’ll benefit greatly in later experiments!

Tags: cell imaging, cellular localization, cellular marker, fluorescence microscopy, fluorescent microscopy, Fluorescent proteins, FPs, FRET

Allele Goes Green!

It’s time for you to jump on the bandwagon with Allele Biotech! We understand the great importance of maintaining the world that we live in, and are excited to participate in this global movement!

San Diego boasts one of the most dynamic biotechnology communities in the world. We’re a close-knit society, and by cooperating and networking with each other, we can make a noticeable difference in our industry. We at Allele are eager to begin working with our customers to reduce, reuse, and recycle!

Read on for a few of the policies that we’re enacting to become more earth-friendly:

Allele uses recycled packaging whenever available. This means that foam boxes and dry ice that we receive are immediately stored for re-use. This cuts down on materials needed and, ultimately, our production costs. This helps to keep our prices low for you! We are also happy to collect used foam boxes and dry ice (and any other re-usable resource) that you may have in your lab, so feel free to leave them with us! Simply pass your re-useable materials to our delivery personnel, and we’ll make use of them – GUARANTEED!

Allele’s company delivery vehicle is a small, fuel-efficient car in order to minimize air pollution. We have worked to develop a meticulously planned delivery route to avoid unnecessary travel.

We plan to employ practices such as electronic invoicing and faxing in order to reduce the amount of paper that we use.

We have and will continue to participate in electronic advertising (for example, e-mail and banner ads) in order to minimize the amount of print advertising used. We have switched our printed advertisements from non-recyclable glossy paper to renewable cardstock.

We are currently researching local vendors to supply our office and laboratory needs, and are implementing recycling services for not only our San Diego branch, but surrounding businesses as well!

Due to our recent acquisition of Orbigen, Inc., we have combined resources and are consolidating workspaces to provide you with a larger product base while reducing the amount of energy used to maintain two operations facilities! In addition, we now have materials and equipment that we are now offered for sale at highly discounted prices – find them here.

Get involved! Allele Biotech is dependent and highly appreciative of our customer feedback. If there’s something you’d like us to work on in our quest to become environmentally friendly, let us know and we’ll put it into practice!

So remember, save money and our earth by buying local at Allele Biotech.

Together, we can create an earth-healthy biotechnology industry!

Tags: biodegradable, energy, environment, green technology, local delivery, lower cost, recycle, recycled material, safety