Archive for December, 2008

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:

Fire and Mello US 6,506,55: RNAi with siRNA >24 nucleotidesr
Tuschl et al. US 108,923 (Tuschl I, pending): synthetic or in vitro produced siRNA 21-23 bps
Tuschl et al US 7,056,704 and 7,078,196 (Tuschl II): synthetic siRNA 19-23, with 3′ overhangs;
Kreutzer-Limmer EP 1,144,623: siRNA 15-21 bps;
Benitec, DNA-driven RNAi DNA driven: granted in 2003, then became under re-examination.

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

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Thursday, December 4th, 2008 RNAi patent landscape 4 Comments

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 36 overlapping oligos of 60 to 80 bases long.
Difficult to do PCR in one piece with all oligos. Switched to 3 separate PCR for about 550 bp each.
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.
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 16 overlapping oligos of 60 to 80 bases long.
PCR in one piece with all oligos.
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.
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.
34 oligos of about 55 bases each direction.
Difficult to do PCR in one piece with all oligos. Switched to 3 separate PCR for about 550 bp each.
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!.
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.
PCR with primers that would introduce restriction sites Not I and Xho I.
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.
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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Thursday, December 4th, 2008 oligos and cloning Comments Off