WO1997007440A1 - Molecular computer - Google Patents
Molecular computer Download PDFInfo
- Publication number
- WO1997007440A1 WO1997007440A1 PCT/US1996/013532 US9613532W WO9707440A1 WO 1997007440 A1 WO1997007440 A1 WO 1997007440A1 US 9613532 W US9613532 W US 9613532W WO 9707440 A1 WO9707440 A1 WO 9707440A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- molecules
- substances
- dna
- computer
- solution
- Prior art date
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06N—COMPUTING ARRANGEMENTS BASED ON SPECIFIC COMPUTATIONAL MODELS
- G06N3/00—Computing arrangements based on biological models
- G06N3/12—Computing arrangements based on biological models using genetic models
- G06N3/123—DNA computing
Definitions
- the present invention describes computing techniques using chemical substances as the computational medium. More specifically, the present invention describes massively-parallel computing techniques using molecules, such as DNA molecules, the computing techniques being carried out using procedures such as cellular processes or experimental processes.
- Computing is a science of calculating some result based on some input information.
- a computer usually carries out processing steps on input information that are used to calculate the result.
- the inventor of the present invention noticed certain similarities between DNA or molecular interactions and the way that a computer operates. He noticed that many of these DNA processes can be carried out in a selective way: that is they will combine in a certain way to the exclusion of other combinations. The inventor had the totally unobvious insight of allowing this analogous operation to be used to steer molecular reactions to actually carry out a computing function.
- a drop of DNA can include more than 10 14 molecules. Each of those molecules has the capability of representing a specific state. Hence that drop can represent more than 10" states.
- the present invention describes how these procedures can be used to carry out multiple operations.
- a substance e.g.. a molecule
- the proper reacting procedure is used which allows selective reacting of the different molecules. This selective reacting uses these molecules to effect complex operations in a massively parallel manner - where each operation can be carried out on all, or at least a large portion of the DNA molecules in parallel.
- a first embodiment of the present invention describes how extremely complicated problems could be done in a few hours using DNA as a computing medium. More generally, however, any molecule or complex (i.e. collection of molecules which are held together by non-covalent bonds) which is capable of reacting in a specified way can be used according to the present invention. All of these features will be described in further detail throughout this
- Another aspect of the invention regards how the different characteristics of this kind of computer are improved.
- the inventor realized that the error rate in such computers is many orders of magnitude higher than that in electronic computers .
- the present invention describes how this hurdle can be overcome.
- Certain intrinsically complex problems such as the directed Hamiltonian path problem and others described herein may be efficiently solved by massively parallel searches. These searches can be organized to take advantage of the characteristics of molecular biology as described herein.
- the techniques of the present invention may be quite well suited for solution of many NP-complete problems.
- the processes are operated to carry out various functions and obtain various results. Importantly, however, the processes are carried out in massively parallel systems, whereby each possible process is carried out many times, in parallel.
- Figure 1 shows a diagram of a simple Hamiltonian path problem
- Figure 2 shows a flowchart of the technique used according to the first embodiment to determine the solution to the problem
- Figure 3 shows a second embodiment of the Hamiltonian path problem
- Figure 4 shows how a Hamiltonian can be encoded as DNA sequences
- Figures 5A - 5C show how DNA representing different states are processed in certain ways
- Figure 6 shows a practical embodiment of the molecular computer
- FIG. 7 shows a flowchart of operation of the
- a procedure is any process that alters properties of chemicals. This includes, for example, a chemical reaction, a chemical process, a cellular reaction, a cellular process, an experimental procedure, an experimental process, a laboratory procedure or a laboratory process.
- a substance is any molecule or a complex of molecules. This includes DNA and other molecules.
- a sticker is a second substance which can be attached to and/or removed from a first substance to modify the information represented by the first substance.
- the first substance might be referred to as a backbone.
- the first embodiment of the invention was directed to solving the so-called Hamiltonian path problem.
- Hamiltonian Path problem can be posed as follows. Assume a fixed number of nodes and possible paths between the nodes. The correct solution to the problem requires determining whether a path can be made which starts at one node, visits all other nodes once and only once, and finally arrives at the last node. Stated more mathematically, given a directed graph G with designated vertices V in and V out . This graph G has a Hamiltonian Path only and only if there exists a sequence of compatible one way edges e 1 , e 2 , e 3 ... The path for a correct solution includes a number of one-way edges must begin at V in , end at V out , and enter every other vertex exactly once.
- a four-node graph with a hamiltonian path will be described with reference to FIG. 1.
- the correct solution to the problem for the four-node graph is trivial.
- the correct solution becomes progressively and exponentially more difficult to solve as the number of nodes increases.
- Step 200 generates all possible paths through the graphs. This is done by assigning each of the nodes to a label, which label is represented by a DNA molecule. This embodiment is carried out using DNA as the preferred molecule. Hence the ith vertex in the graph is associated with a DNA molecule which is denoted as O i .
- This first step obtains the universe U including every possible path through the directed graph.
- the remainder of the process consists of determining criteria that can be used to identify all incorrect paths representing the incorrect solutions, and discarding those paths until only the correct path representing the correct solution, if any, remains.
- Step 204 discards all paths that have any number of vertices other than N.
- U 2 includes paths staring at V in and ending at V out , and which enter the proper number of nodes.
- Step 206 discards everything that does not enter every vertex of the graph. The result is the solution S. If S has any DNA in it, then the graph has a Hamiltonian path represented by the molecules which have survived the test. If not, there is no solution to the Hamiltonian path problem.
- one important feature of this embodiment is its ability to generate many, many molecules, each of which represents some part of the problem. Since there are so many molecules generated, it becomes
- each possible solution is assigned to a designated sequence of DNA.
- DNA is a double-stranded molecule in which one strand of DNA is annealed to a second strand of DNA (i.e., the "complement").
- the strands of DNA are polar in nature, with a phosphate group at the 5' end, and a hydroxyl group at the 3' end.
- Each nucleotide has a so-called Watson-Crick Complement, which determines whether two strands of DNA can anneal to each other.
- the nucleotide A in one strand of DNA will only anneal to the nucleotide T in a second strand of DNA.
- nucleotide C will only anneal to the
- nucleotide G An example of a double-stranded molecule is shown here with its phosphate and hydroxyl groups.
- the Watson-Crick complement of ATTCG is TAAGC.
- DNA's chemical composition allows a first stand of DNA to anneal to a DNA molecule which is the "complement" of the original compound. DNA molecules usually only anneal to other DNA molecules which contain their complement.
- the present embodiment first obtains a universe of all possible paths. This is done by obtaining a unique DNA molecule for each part of each path, and then combining those parts to get each possible path.
- Each vertex in the graph is assigned to a particular DNA sequence O.
- the DNA sequence is defined as including a forward portion O f and a rear portion O r .
- FIG. 1 shows four vertices A, B, C and D.
- Vertex A is assigned for example to the 6-mer 5'AGTCAT3'. The 6-mer sub-divides this DNA molecule into A f and A r , where A f is AGT and Al is CAT.
- B, C and D are assigned with DNA sequences: B is assigned 5'TTGACG3' where TTG is B f and ACG is B r .
- edge from A to B is defined as the edge including the last part of the first vertex (A r ) and the first part of the second vertex (B f ).
- A-B is encoded as A r B f ; here 5'CATTTG3'.
- an oligonucleotide O I ⁇ J is created. If the oligos are x mers, then the oligonucleotide O I ⁇ J is the 3' x/2-mer of O I followed by the 5' x/2-mer of O J . This construction preserves all edge orientation. For example, O 2-3 will not be the same as O 3-2 .
- step 1 of the algorithm generating all possible random allowable paths through the graph.
- Each such DNA molecule representing every edge between vertices of the graph represent every allowable transition and hence each possible solution. Moreover, there are multiple copies of each such DNA molecule. Every DNA molecule representing every edge, that is, for example, DNA molecule 1 representing A-B, DNA molecule 2 representing B-C, and so on is included in the mixture.
- the next step is to bind together all possible compatible edges. Accordingly, we also add the complement of each original vertex. For example, (A not) will bind together A f to A l , thus binding all paths that end with A f to all paths that begin with A l .
- A is 5'AGTCAT3'
- B is 5'TTGACG3'
- C is 5'GATTAC3'
- (B not) is 3'AACTGC5'. Hence the 3' part of (B not) will complement or bind to the B f in A ⁇ B edge.
- A-B is 5'CATTTG3' (A l B f ); B ⁇ C is 5'ACGCAT3' (B l C f ).
- B not equals 3'AACTGC5'.
- the 3' part of the (B not) molecule, (B f not) will anneal to the B f in the A ⁇ B edge .
- the 5 ' part of the (B not) molecule , (B l not) will bind to the B l in the B-C edge.
- the vertex nots effectively link or connect together the molecule representing A-B to the molecule representing B ⁇ C. It will bind together these edges to form eventual larger, or combinatorial, molecules.
- This reaction continues to form a plurality of DNA complexes that are longer in sequence. If there are
- these sequences will represent every possible path formed between the vertices . That is, all random allowable paths through the sequence will have been generated at step 200.
- DNA ligase is used to create a covalent bond between the 5' end of one DNA molecule and the 3' end of another DNA molecule.
- Ligase is an enzyme that repairs breaks in DNA and can connect two DNA molecules end to end.
- the ligase ensures that the molecules representing A ⁇ B and B ⁇ C, etc., are covalently connected end to end to become a single molecule.
- the vertex nots making up the other strand of DNA are covalently/chemically connected end to end by ligase.
- the ligase thus creates two (one for each DNA strand) long continuous chains of DNA that can withstand the further processing that is necessary to eliminate the molecules representing the undesired paths.
- Step 202 requires discarding everything that does not start with v in and end with v out .
- the operation according to the present embodiment uses an amplification technique to narrow down the proper parts.
- a polymerase chain reaction (“PCR") is preferably used to selectively reproduce molecules in a particular tube.
- PCR polymerase chain reaction
- the primer is complementary to one of the desired ends.
- This primer pair reproduce any molecules that have the proper ends for example 10 6 times.
- Step 204 discards all paths that do not enter exactly n vertices, where n is the number of vertices.
- embodiment will discard sequences which are too long or too short. This operation is carried out using gel
- Electrophoresis is a process of using electronic separation to cause migration of molecules through a matrix. Electrophoresis puts all the molecules at one end of a gel medium and exposes them to an electric field. The short DNA molecules move more quickly under the electrophoretic field, and long DNA molecules move more slowly. This forms an eventual product with bands of nucleotides, where the final positions bands in the gel are proportional to the number of nucleotides in the sequence.
- the size discrimination according to the present invention includes viewing the bands, and cutting out and discarding all those bands which are too short or too long. If necessary, this process can be performed with a high degree of resolution, permitting discrimination between molecules that differ by only a single nucleotide.
- U 2 as formed from step 204 has start and end portions which are correct, and all sequences which are the right length. What is left includes the correct Hamiltonian path and incorrect paths which have skipped one or more vertices and hence visited others more than once.
- the last step 206 is hence used to keep only those paths which enter each vertex of the graph once.
- the preferred mode of this invention carries this out by doing affinity separations.
- Affinity separation is preferably carried out using lum beads with material
- agarose beads which have a streptavidin binder thereon.
- Streptavidin has a high affinity for biotin, a ligand that can be attached to the end of a DNA molecule.
- biotin is attached to (B not), and the biotin/(B not) conjugate is linked to the streptavidin binder of the agarose beads.
- prepared beads can then capture DNA molecules that contain B, the complement of (B not).
- the preferred mode of this embodiment therefore associates each vertex i in the graph randomly with a 20-mer sequence.
- 50 pmol of (O i not) and 50 pmol of O i ⁇ j are mixed together in a single ligation reaction.
- Each oligonucleotide (50 pmol) with 5'-terminal phosphate residue, 5 units of T4 DNA ligase (Boehringer-Mannheim, Germany), ligase buffer, and ddH 2 O to a total volume of 100 ⁇ l was incubated for 4 hours at room temperature.
- the (O i not) oligonucleotides serve as splints to bind oligonucleotides associated with compatible edges together for ligation.
- Figure 4 shows the assignment and ligation reaction.
- a random 20-mer oligonucleotide is generated for each vertex.
- Figure 4 shows only O 2 , O 3 and O 4 .
- Each edge in the graph is effected by an oligonucleotide O i ⁇ j from the 3' 10-mer of O i and the 5' 10-mer of O j .
- Figure 4 shows the sequences O 2-3 for edge 2-3 and O 3-4 for edge 3-4.
- Figure 4 shows all oligonucleotides written as 5' to 3' except for (O3 not). This results in the formation of DNA molecules encoding random paths through the graph.
- Step 202 is carried out by amplifying using PCR with primers O 0 and (O 6 not). Only those molecules that begin with vertex 0 and end with vertex 6 are amplified. All PCR amplifications were performed on a Perkin-Elmer (Norwalk, CT) 9600 thermal cycler. For amplification in Step 2, 50 pmol of each primer and 5 units of Taq DNA polymerase
- Step 204 is run on an agarose gel, with a 140-base pair (bp) band, corresponding to double-stranded DNA having 7 vertices.
- the DNA corresponding to paths entering exactly seven vertices is excised and soaked in doubly distilled water and then gel-purified several times to enhance its purity. All gels were 3 to 5% agarose (NuSieve, FMC Bio-Products, Rockland, ME) in tris-borate-EDTA buffer with ethidium bromide staining
- Step 206 finally affinity-purifies the newest sample U2 with a biotin-avidin magnetic beads system. This was accomplished by a two-step process.
- step A the magnetic beads system was used to generate single-stranded DNA from the double-stranded DNA product of Step 3. (O 6 not)
- oligonucleotides were 5' biotinylated with LC Biotin-ON Phosphoramidite (Clontech).
- the product from Step 3 was amplified by PCR with the use of primers O o and biotinylated (O 6 not).
- the biotin group on the amplified product allowed the double-stranded DNA to be annealed to streptavidin group on the paramagnetic particles (Promega, Madison, WI) by incubating in 100 ⁇ l of 0.5 ⁇ saline sodium citrate (SSC) for 45 min at room temperature with constant shaking.
- SSC 0.5 ⁇ saline sodium citrate
- Particles were washed three times in 200 ⁇ l of 0.5 ⁇ SSC and then heated to 80°C in 100 ⁇ l of ddH 2 O for 5 min to denature the bound double-stranded DNA.
- step 3 corresponding to the product of step 3 was retained.
- step B the magnetic beads system was used to affinity purify DNA containing 0 1 .
- the product is amplified by PCR and run on a gel.
- Figures 5A-5C show the results of these procedures.
- lane 1 is the result of the ligation reaction in Step 200.
- the smear with striations is consistent with the construction of molecules encoding random paths through the graph of Figure 3.
- Lanes 2 through 5 of Figure 5 show the results of the PCR reaction in Step 202.
- the dominant bands correspond to the amplification of molecules encoding paths that begin at vertex 0 and end at vertex 6.
- Figure 3B shows the results of a "graduated PCR” performed on the single-stranded DNA molecules generated from the band excised in Step 204.
- Graduated PCR forms a "print out” of the results and is performed by running six different PCR reactions with the use of O 0 as the right primer and (O i not) as the left primer in the ith tube.
- O 0 the right primer
- O i not the left primer in the ith tube.
- graduated PCR will produce bands of 40, 60, 80, 100, 120 and 140 bp in successive lanes.
- Fig. 5B The most prominent bands in Fig. 5B are those that would arise from the superimposition of the bands predicted for the three paths described above.
- Step 3 corresponding to path 0 ⁇ 1, 1 ⁇ 3, 3 ⁇ 4, 4 ⁇ 5, 5 ⁇ 6 were not expected and suggest that the band excised in Step 3 contained contamination from 120-bp molecules. However, such low weight contamination is not a problem because it does not persist through Step 206.
- Figure 5C shows the results of graduated PCR applied to the molecules in the final product of Step 206. These bands demonstrate that these molecules encode the Hamiltonian path 0 ⁇ 1, 1 ⁇ 2, 2 ⁇ 3, 3 ⁇ 4, 4 ⁇ 5, 5 ⁇ 6.
- Step 206 the magnetic bead separation, was the most labor-intensive, requiring a full day at the bench.
- the number of procedures required should grow linearly with the number of vertices in the graph.
- the labor required for large graphs can be reduced by use of alternative procedures, including the automation technique described herein with reference to Figure 6. Less labor-intensive molecular algorithms can also be used.
- the number of different oligonucleotides required also increases linearly with the number of edges.
- the quantity of each oligonucleotide needed is a rather subtle graph theoretic question. Roughly, the quantity used should be just sufficient to ensure that during the ligation step 200, a molecule encoding a Hamiltonian path will be formed with high probability if such a path exists in the graph. This quantity should grow exponentially with the number of vertices in the graph.
- Step 206 leaves a solution with molecules representing all Hamiltonian paths for the problem.
- the graduated PCR at the end of step 206 will produce a superimposition of the bands corresponding to all of these Hamiltonian paths in the n-1 successive lanes.
- Step 206 there is a solution in the chemistry sense containing molecules encoding all Hamiltonian paths for ⁇ G, v in ,v out >.
- the graduated PCR performed at the end of Step 206 will produce the superimposition of the bands corresponding to all of these Hamiltonian paths in the n - 1 successive lanes. For some lane i, a band of least weight (40 bp) will appear. This indicates that some Hamiltonian path begins with v in and proceeds directly to vertex i.
- One simple error correction technique for this embodiment would include using a conventional electronic computer to confirm that a putative Hamiltonian path actually occurs in the graph at the end of any calculation.
- This satisfiability problem includes 4 variables, A-D, and 4 connectives (ands and ors).
- the satisfiability problem consists of determining if there is a possible solution, e.g., a truth - assignment, for which ⁇ comes out true.
- a possible solution e.g., a truth - assignment
- there are 2 4 16 truth-assignments. Each one gives ⁇ as being true or false, and ⁇ is satisfiable if and only if at least one truth-assignment gives ⁇ true.
- the preferred 700-mer will be of the form for example O5'T 1 F 2 ...O3' where T 1 F 2 ... is a 700-mer.
- the totality of 700-mers would require approximately 0.93 pounds of DNA, which is about the amount of DNA in the human body. Since there is so many possibilities, we can postulate that each one of the DNA molecules, encoding each of the possible solutions, is contained within that universe. This universe is appropriately tested as above, to leave only those solutions which match the predetermined criteria, i.e.
- An important feature of the present invention is its ability to operate in a massively parallel way and with enormous energy efficiency.
- Cellular evolution has optimized energy efficiency processes in the cells.
- a typical desktop computer can execute approximately 10 6 operations per second.
- the fastest supercomputers currently available can execute approximately 10 12 operation per second.
- test tube For automation purposes described herein, it is convenient to define a test tube as being a set of molecules of DNA or more formally a multi-set of finite strings over the alphabet A,C,G,T. It is of course possible that in real systems the time and accuracy of a separation may depend on the tube and symbol being considered. However, for convenience it will be assumed that a uniform time and accuracy can be given.
- +(T,S) is all of the molecules of DNA in T which do contain the consecutive subsequence S.
- -(T,S) is all of the molecules of DNA in T which do not contain the consecutive subsequence S.
- Step 202 in the first embodiment can be thought of a separating steps, since it requires determining which molecules do have specific beginning and end sequences.
- This second embodiment uses a more structured language of programming. Tubes are received as input. The output is returned as yes, no or a new tube or set of tubes.
- the following program shows inputting a tube, and returning "yes", if and only if the tube contains as sequence which is composed entirely of A.
- This program assumes a four element alphabet: A, G, T and C.
- T2 -(T1, G)
- T3 -(T2, T)
- the last step can be changed to OUTPUT (T3), to
- This second subset of the second embodiment preferably uses a restricted model , which has different characteristics which may make it more practical to implement.
- the amplify operation used in the first embodiment may be one of the more difficult operations to actually carry out Amplification is a complicated and rare process.
- amplification is most often applied to special biological molecules such as DNA and RNA, and certain living things.
- Amplification requires construction and processing of covalent bonds.
- One objective of this second embodiment is to avoid or restrict the use of amplification, and hence to allow increasing the size of the alphabet.
- One advantage of obviating the amplification is the ability to use substances other than DNA.
- DNA has a natural structure allowing the occurrences of certain symbols to be monitored, and to speak of sequences, this may not be true for other types of substances.
- groups encoding the symbols in other types of substances may simply be placed anywhere on a standard and potentially non-linear molecular backbone.
- the elements of a "tube" are not, therefore, limited to DNA sequences. More generally, these elements might be called aggregates. These aggregates, in general, are subsets of ⁇ . Separation will be allowed with reference to symbols only.
- the tube is a multiple set of aggregates over an alphabet ⁇ . Given a tube, one can perform the following operations. 1. Separate. Given a tube T and a symbol s ⁇ , produce two tubes +(T,s) and -(T,s) where +(T,s) is all of the aggregates of T which contain the symbol s and -(T,s) is all of the aggregates of T which do not contain the symbol s.
- each vertex in the graph can be colored either red, green or blue in a way such that after coloring, no two vertices connected by an edge have the same color.
- the problem must first be posed in a way that allows the problem to be solved by selectivity in molecular operations.
- the elements of the input tube T are in one to one correspondence with the universe of possible ways to assign colors to the vertices of G.
- the program After 5k separations, 2k merges and 1 detect, the program will output 'yes' if G is 3-colorable and 'no' otherwise.
- the restricted program answers the 3-colorability problem for the graph G in 'linear' time based on the number of edges.
- An electronic computer in contrast, would require
- This technique can be used to efficiently solve the well known (NP-complete) 'satisfiability problem': given a propositional formula (not necessarily in conjunctive normal form) decide if it is satisfiable.
- Merging can be done by pouring the contents of the multiple input tubes into a single tube. This is much faster and less error prone than separating. For many of the problems attacked by these restricted computers, detect is done rarely.
- biomolecules may have several advantages.
- a molecular computer for satisfiability could create the input tube T consisting of the molecules encoding all possible truth assignments just once. Then given a formula ⁇ , T would be used to determine whether ⁇ was satisfiable. Once that computation was completed, the tube T would be recreated and the next formula processed.
- Such a system would encode an aggregate in approximately one fiftieth of the mass used by the DNA computer. Hence it would provide approximately 50 times the parallelism per unit size. Further, fullerenes are quite stable.
- the restricted model of molecular computation given above in the first and second embodiments is memoryless in the sense that the molecules themselves do not change in the course of a computation.
- the state of the computation is represented primarily by the collection of the molecules - i.e., which molecules exist in any tube.
- the present embodiment describes a system using a molecular computer with memory.
- the molecules in the solution can be altered. This alteration causes the molecules to represent stored information.
- This model has the same power as the unrestricted model, but also also allows storing results of interim computations, i.e., storing results of different tests.
- This model is used as a memory of a computer.
- a tube is a multi-set of aggregates over an alphabet
- ⁇ ⁇ a 1 ,a 2 , ... ,a n ,b 1 ,b 2 , ... ,b n ⁇ .
- Each location l therefore acts like a memory element.
- T' ⁇ (-(T,a 3 ),-(T 1 b 5 ))
- each a i is associated with some small functional group and b i is obtained from ai by the non-covalent attachment of a small molecule which binds to a i .
- This above-described operation essentially records a history in the working molecules.
- the present embodiment suggests a technique of using molecular stickers to record this history.
- the existence of a sticker at each sticker position may indicate whether a material has been through a particular computation, or has not been through that computation. More generally, however, the presence or absence of the sticker is used to determine something about the computation.
- Each sticker can be embodied as a DNA sequence.
- Each sticker of course, has an inherent complement (s 1 not), (s 2 not) ... (s 10 not). These complements will be called
- a sticker evaluating molecule can include a complex of locations. Each of the locations is either complemented by a sticker or not. The presence of the sticker indicates that the molecule has been tested for the particular subproblem.
- a plurality of sticker locations are placed at the end of the molecule. If a sticker location is complemented with the sticker, the condition associated with that location is evaluated as a "1". If a sticker location is not
- an operation can be marked by attaching sticker S 5 to its appropriate locaton. Later, all molecules can be run across bead-bound S 5 . Only those molecules which are not "marked” with S 5 will stick.
- the sticker evaluating molecule could be the genome of a virus, e.g., the M13 virus.
- the molecule could be the genome of a virus, e.g., the M13 virus.
- non DNA e.g. fullerenes.
- the stickers can be any substance which reacts with the sticker evaluating molecule in a predictable way that can be later detected.
- the detection can use separations, for example.
- the seperations can be done based on whether stickers are present.
- the generalized molecular computer can also be used to embody the memory model.
- a i is associated with an oligonucleotide O i and b i is associated with a methylated version of O i .
- EcoR I methylase will catalyze the transfer of a methyl group (from S- adenosylmethionine) resulting in 5' ...GA m ATCC...3'.
- EcoR I methylase will not methylate oligos without the subsequence GAATTC. Other techniques can be used to demethylate and hence clear the stickers.
- This third subset provides renewable and reusable workspaces.
- This embodiment uses two kinds of stickers - weakly bound stickers and strongly bound stickers.
- the weakly bound stickers can be removed without affecting anything about the strongly bound stickers.
- any of the techniques described above are used to provide a plurality of strong bits.
- evaluating molecule workspace w is formed of weaker sticker bits. These weak bits can be dissociated from their bonds by heating. According to this technique of the present invention, the strong bits are used to store information or to mark various paths through the operation as described in the previous embodiments. These weakly-bound stickers are used as working memory. The contents of this working memory can be cleared by heating the solution of the substances. The workspace is heated up sufficiently to dislodge these weak stickers but not to dissociate the strong stickers from the sticker evaluating molecule. Fourth Subse t
- An alternative memory model facilitates the separation of various different particular elements.
- This memory model uses a sequence M of a length of, for example, 10,000.
- the active sequence for M is not so important as the requirement for many copies of M.
- This embodiment suggests using the genome of a phage, e.g. a bacterial virus.
- the phage can be "fed” to form the copies of M. All these copies of M are the same.
- M includes a plurality of subparts which we call B1,
- the memory of this embodiment has 200 locations; i.e. it is a 200 bit memory.
- Each of the subparts is a block of length 50. Each block corresponds to a location in the memory.
- Each block is broken into three parts, Z, C and O.
- Z and O are 10 mers, while C is a 30 mer.
- the portion Z represents 0, O stands for 1, and C represents common.
- the locations in memory are written as follows: the present embodiment uses stickers which are oligos. Each block B i described above has two different sticker oligos.
- the sticker oligo (Z i C i not) represents an indication that the block represents a 0 (that is Z is complemented).
- the sticker (C i O i not) indicates that the block represents a 1.
- Location i can be set to 0 by annealing sticker (Z i C i not) to M. Location i can be set to 1 by annealing (C i O i not) to M.
- An advantage of this system is the facility with which the differently-coded elements can be separated.
- This string has location B5 annealed to sticker (Z 5 C 5 not).
- the 40 base pairs of B5 include Z 5 and C 5 being paired.
- the 10 base O 5 is unpaired.
- those that have B 5 set to 0 will anneal to the bead-bound (O 5 not).
- other strings that have location B 5 set to 1 will have location B 5 annealed to (C 5 O 5 not).
- the O 5 is not exposed.
- Those strings will not anneal to bead-bound (O 5 not).
- the molecules in this way can be run over bead-bound (O 5 not). Only those molecules that have location 5 set to 0 will be captured on the beads.
- the captured strings include those with location 5 set to 0. These can be released by eluting. Preferably this is done by heating to a eluting temperature. This temperature will break the 10-base pair bond between the O 5 and the bead-bound (O 5 not). This eluting temperature is not high enough to break the 40 base pair bond between B 5 and (Z 5 C 5 not), or any other stickers elsewhere on the molecule.
- Each block B ⁇ consists of X i Y i Z i .
- a sticker oligo (X i Y i Z i not). This requires only 200 sticker oligos, one for each of the 200 blocks. Again, the sticker oligos are short and can be synthesized easily.
- Any location i is set to 1 by annealing sticker (X i Y i Z i not) to M.
- the location i is set to "0" when no oligo is annealed thereto.
- bead-bound (Y i not) will catch all molecules M which have Y i exposed. In other words, those molecules with position 5 that are covered will not be caught by beads which have bead bound (Y 5 not). Those molecules can be captured by washing the beads at low temperatures. However, those molecules which do not have position 5 covered will be caught by those beads.
- the captured strings can be released by eluting at a temperature sufficient to break the 10-pair base bond, but not hot enough to break the 50 pair base bond.
- This fifth subset is simpler, since it requires less stickers be synthesized. However, it is not possible to use this system to detect improper states, i.e. those states which do not fall into either positive or negative.
- This embodiment describes a DNA-based implementation of the 'restricted model' as described in the third embodiment.
- the alphabet of symbols ⁇ as used herein contains two special symbols s 5 , and s 3 ,. Each symbol S ⁇ ⁇ is associated with an oligonucleotide ("oligo"), i.e. a short sequence of nucleotides.
- oligo oligonucleotide
- These conditions include, for example, control of temperature, pH, salt concentration or ion concentration.
- a Merge of tubes is accomplished by pouring the contents of all tubes to be merged into a single tube.
- a Separate is carried out on a tube T with respect to a symbol s as follows. If s is associated with the oligo O, a separator which consists of molecules of the oligo (O not), the Watson-Crick complement to O conjugated to solid
- supports are used.
- the magnetic bead system described in the first embodiment can be used, or an
- a Detect on a tube T uses a PCR with primers
- This new model can be used, for example, to solve an instance of the 'satisfiability problem'.
- ⁇ ⁇ T i
- i 1,2,...,70 ⁇ ⁇ ⁇ F i
- i 1,2,...,70 ⁇ ⁇ ⁇ s 5' ,s 3' ⁇ , with a 'true' symbol and a 'false' symbol for each of the variables and the required 'PCR symbols' s 5' and s 3' .
- Each symbol of ⁇ is associated with a randomly chosen
- oligo associated with T i O i T
- oligo associated with F i O i F
- oligos associated with s 5' and s 3' O 5' and O 3' respectively.
- the input tube T includes the whole universe of all possible truth assignments for the problem.
- the input tube consist of the set of all DNA
- T is preferably created as follows. First, all of the
- Each bead is approximately 1 micron in diameter and has a mass of approximately 1.3 pg.
- Each bead can bind approximately 7.8 ⁇ 10 5 biotinylated oligos. Assuming that one bead-associated biotinylated oligo will bind one target molecule, then binding 2 69 target molecules (the number of targets in the largest separation done during this computation) would require approximately 984 g (approximately 2.2 lb.) of beads with a volume of roughly 0.39 liters (approximately 0.49 qt.) These beads would have a volume of approximately 750 ml.
- Each incarnation of a restricted computer is associated with the parameters ⁇ , time for operation; ⁇ +, the
- the DNA computer described in the second embodiment could carry out the separation as follows:
- Anneal Incubate the contents of T with the beads and allow time for molecules with the correct consecutive subsequence to anneal. Pour off the liquid phase containing molecules that do not anneal.
- T 1 +(T,s) and T' 1 - -(T,s).
- This tube should ideally have the 'winners', i.e.
- the tube T1W is used as the input to an entire rerun of the whole computation to re-test all of the purported 'winners'.
- the new tube T2W includes all of the molecules which went through the entire computation twice and both times ended in the winning tube.
- the probability of the materials in the tube T2W being incorrect is therefore greatly decreased. That process can be repeated through r runs of the entire computation to make tube TrW, which will contain two types of molecules. TrW contains the 'true winners' those that really do encode satisfying truth assignments and the 'false winners' those which encode unsatisfying truth assignments - but somehow, through errors, made it into TrW.
- a 'false winner' must have at least one error on each pass through the computation.
- Tw10 is at most 1/13.5 (less than one). Hence, even if ⁇ is not satisfiable, it is unlikely some 'false winner' will end up in Tw10. Now P 10 good is approximately 0.94. So even if there is just one satisfying truth assignment for ⁇ , there is at least a 94% chance the molecule encoding that
- the basic system of the present invention keeps DNA samples in tubes to follow the tube model given above.
- Each tube includes a small sample of some working substance.
- Each tube is numbered, and may include an optically-detectable indicia thereon, such as a holographic indicia or a bar code.
- Step motor is driven by pulses from processor 614 which controls the overall operation according to a pre-stored flowchart.
- the processor also controls other operations as described herein.
- Each of the tubes 600, 602 includes a sample of substances ⁇ in this embodiment most samples are DNA.
- Different tubes may contain different samples.
- processor stores information indicating the position of tubes in the carousel and the position of the carousel itself. For example, there is a specified position on the carousel shown as position 604. That position might represent the zero position. By knowing the current position of that location, the processor can always
- Tube 600 can be rotated to the position shown as Y. Alternately, or additionally, the indicia on the tube can be read to ensure that the correct tube is being accessed.
- Each of a plurality of robots 620 includes a robot arm 624.
- the robot arm reaches in to the carousel at position Y, or some other programmable location, to grab the tube at the position Y.
- Each of the robot arms can grab a tube from a different group of locations.
- Each of the robot arms also includes an associated work area 630 in which the grabbed tube can be further processed, e.g. by sperating merging, adding stickers, eluting or other operations as described herein.
- Figure 6 shows the robot arm in a second position with the tube S 10 therein.
- the tube S 10 has its contents poured over apparatus 632.
- Apparatus 632 can be, for example, magnetic beads which are marked with a complement of a particular element.
- the contents in tube 632 have been removed and held by robot arm 622.
- the contents of S 10 are poured over the apparatus in 632. Every element which has a 0 in the tenth bit, for example, may be separated out by the element 632. Accordingly, the content of the tube 634 includes all materials that do not have a 0 in the tenth bit.
- Tube 634 may then be grabbed by robot arm 628 of robot 626 and returned to the carousel to a specified location. The returned tube 634, therefore, includes only materials which do not have 0 in the tenth bit.
- each robot operation pulls a tube from any position within the reach of the robot.
- the tube is then further processed in the workspace 630 with other materials which may be taken from other tubes, to carry out some different operation as described above.
- Each of these robot operations can be accomplished in approximately one minute, thus increasing the previous estimate of operations per unit time by another order of magnitude. Moreover, many robot operations can be done in parallel by different robots, to further decrease the necessary time of operation.
- the carousel embodiment preferably uses a plurality of tubes around the carousel with a number of bead operators. Some tubes outside the carousel can be used as separators, merges and sticker manipulators. A carousel having 3142 -1/4 inch tubes would be about 10 feet in radius. While this embodiment describes operation using robot arms, it should be understood that the key point here is the ability to move the molecules from a first position to a second position. Other means of moving these molecules, including pumps, electrostatic movements, and other similar features could also be used.
- the flowchart of operation of the apparatus of Figure 6 is now described with reference to the flowchart of Figure 7.
- the processor 614 carries out various operations.
- the system detects the next operation, determines the tube locations required for that next operation, and command the robot arms to grab those tubes . This requires that the processor control the position of the carousel 610, and the movement of the robot arms between their different positions. The tubes are then located into the workspace 630.
- the system carries out the desired operations on the tubes which have been obtained in this way.
- Step 704 is optional, since it requires the processor to look ahead to try to define the appropriate locations for these tubes which will enable them to be most-efficiently obtained during a subsequent operation.
- Algorithms for such pipelining are well-known. For instance, if a problem can be decomposed as described above, different parts of the problem can be simultaneously computed.
- step 706 the materials are replaced in the carousel, and the process may continue.
- the solution that is determined by the computer may be tested using an electronic computer at step 708.
- the advantage of the electronic computer carrying out the testing is as follows. Say for example there are 10 22 possible solutions to a problem, and each one might take even a microsecond for an electronic computer to calculate. This still yields 10 16 seconds (3.1 ⁇ 10 8 years) to solve the problem.
- This test operation combines the advantages of electronic and molecular computers, and may in some cases obviate the need for error correction described above.
- the goal of the experiment was to find a molecule of
- RNA which would ligate two substrate molecules of RNA ⁇ 1 and ⁇ 2 ( ⁇ 1 and ⁇ 2 were base pair complementary in such a way that hybridization would bring their 5'-triphosphate and 3'-hydroxyl groups into proximity in preparation for ligation).
- a pool of approximately 4 25 random sequences of RNA was developed. Each RNA in the pool was bound to a copy of ⁇ 2 at the 5' end and a copy of a constant region C at the 3' end.
- a tube containing approximately 4 25 molecules was created, and each molecule in the tube had the form ⁇ 2 RC, where R was a some molecule of RNA from the original pool. To this tube an excess of ⁇ 1 was added.
- the 'winner' (actually 'winners') of Bartel and Szostak experiment is not a true enzyme, since it is 'used up' in acting on its substrates.
- the value of having the 'winner' RNA 'anchored' to one of the substrates is that once the winner acts, it. becomes permanently anchored to the reaction product. This distinguished it from the rest of the RNA pool and allows it to be retrieved for later identification. If one wished to create a true enzyme, one might have to identify the 'winner' despite the fact that after acting on its substrate it reenters the pool.
- At least one (and perhaps more than one) of them will contain a 'winner'. If P C contains a 'winner', then we know some 'winner' begins with C. Next we repeat the experiment with P CA , P CC , P CG and P CU . Again at least one must contain a 'winner'. Say P CA , then we know some winner beings with CA. Continuing in this fashion we will come to know the exact sequence of some 'winner' as desired.
- the present inventor recognized the similarity of this technique to that used for demonstrating that if there exists a polynomial time algorithm for deciding
- RNA in the pool could also have fixed constant regions if that was desired.
- the system is heated then cooled. This randomizes the stickers. After the randomize operation, all desired representations are obtained, up to the limit of statistical variability.
- the other half of the virgin primary strands are then added, and heated to apply the randomize operation.
- This randomization operation can be used to prepare initial strands for the above discussed embodiments.
- Point mutation is an operation to allow some statistical variability in the process. The variability is carried out by allowing certain ones of the stickers to fall off from each primary strand.
- a partial randomization operation would cause 1 in 50 stickers to fall off from each primary strand at random. These stickers could then stick to new primary strands at random. Each of the primary strands would therefore randomly loose a few stickers and randomly pick up a few new stickers. This allows sticker mutation without enzymes or replication, and allows efficient implementation of
- weakly bound stickers can be formed. These weakly bound stickers include stickers which differ from standard stickers in that they can be made to dissociate from the primary strand more easily. For example, these weakly-bound stickers dissociate at lower temperature or with different salt concentration. For example, weakly bound stickers can be created by incorporating mismatches, decreasing length or using backbones other than phosphodiester. Similarly DNA oligos can be used as weakly bound stickers in a system where standard stickers bind tighter. For example when standard stickers are longer or use other backbones, for example peptide backbones. This randomizes only the weak stickers, and allows the normal stickers to remain in place. It is also possible to remove weak stickers entirely while leaving standard or more strongly bound stickers in place.
- Ninth embodiment Data representations and translation between the data representations.
- the above embodiments have described storing data, including computing states and memory states, and two different ways of representing these states using molecules.
- the "strand" representation such as described in the first embodiment, represents the states using strings of molecules which are held together.
- the kinds of the molecules in the solution represents the current state of the computation and problem.
- This representation has many advantages, including the ability to separate by various techniques, and to clone by PCR.
- the sticker representation is described, for example, in the third embodiment.
- the sticker representation can be used for a memory, or to represent states.
- the information content of the unit can be easily changed, but there are no straightforward techniques to clone the units.
- the present system facilitates using both representations during a computation .
- Each 50 mer block B i is represented as L i M i R i , where L i is a 20 mer, M i is a 10 mer, and R i is a 20 mer.
- a tube of molecules that remains after a computation includes the primary strand, and some subset of stickers attached. A different subset may be attached for different complexes.
- Each block B i in each complex has now attached to either an S i or an S' i .
- the condition of the block depends on whether the block originally had an S i attached or not. It is apparent that those blocks with S i attached will not receive S' i .
- Ligase is added to covalently bond the sequence of stickers and substitute stickers to form a single secondary strand.
- the primary strands are then removed.
- These secondary strands are now strand representations of the same information that was represented by the sticker strands from which they were derived.
- the correspondence shows that B i in the secondary strand has M i if the complex from which it was derived had a sticker at B i .
- the block B i on the secondary strand has M' i if the complex from which it was derived did not have a sticker at B i .
- These secondary strands may be cloned using PCR, and hence include many of the advantages of the strand
- An alternative embodiment immediately adds polymerase and an excess of universal nucleotide monomers to the original tube of complexes. These universal nucleotides complement all of the ATCGs, and hence fills in the gaps between stickers.
- the molecules, DNA, and other substances described herein are merely examples of the possible substances which might find application with the present invention.
- research in molecular biology may provide improved techniques for manipulating macromolecules .
- Research in chemistry may allow for the development of synthetic designer enzymes.
- a single molecule of DNA can also be used to encode the "instantaneous description" of a Turing machine.
- Currently-available protocols and enzymes could be used to induce successive sequence modifications, which would correspond to the execution of the machine.
- Single molecules can be detected in solution by labeling them with fluorescent ligands as described in Eigen et al, "Sorting single molecules", PNAS 91:5740-5747 (June 21, 1994).
- Physicists have also determined ways to detect a single subatomic event in a huge volume of space when detecting neutrinos. This could also be used for the detect operation.
- a molecular computer could also use a Describe
Landscapes
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Theoretical Computer Science (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Mathematical Physics (AREA)
- Nanotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Chemical & Material Sciences (AREA)
- Evolutionary Biology (AREA)
- Computational Linguistics (AREA)
- General Health & Medical Sciences (AREA)
- Artificial Intelligence (AREA)
- Biomedical Technology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Data Mining & Analysis (AREA)
- Evolutionary Computation (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Computing Systems (AREA)
- General Engineering & Computer Science (AREA)
- General Physics & Mathematics (AREA)
- Software Systems (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU67801/96A AU6780196A (en) | 1995-08-21 | 1996-08-21 | Molecular computer |
JP50957197A JP2001515614A (en) | 1995-08-21 | 1996-08-21 | Molecular computer |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US254895P | 1995-08-21 | 1995-08-21 | |
US55600595A | 1995-11-13 | 1995-11-13 | |
US08/556,005 | 1995-11-13 | ||
US60/002,548 | 1995-11-13 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1997007440A1 true WO1997007440A1 (en) | 1997-02-27 |
WO1997007440A9 WO1997007440A9 (en) | 1997-06-26 |
Family
ID=26670532
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/013532 WO1997007440A1 (en) | 1995-08-21 | 1996-08-21 | Molecular computer |
Country Status (3)
Country | Link |
---|---|
JP (1) | JP2001515614A (en) |
AU (1) | AU6780196A (en) |
WO (1) | WO1997007440A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001059704A1 (en) * | 2000-02-11 | 2001-08-16 | Universiteit Leiden | A biological molecule based computing method based on a blocking principle |
EP1154376A1 (en) * | 2000-05-12 | 2001-11-14 | Universiteit Leiden | The use of proteinaceous molecules in methods for molecular computing |
WO2004068398A1 (en) * | 2003-01-30 | 2004-08-12 | Fujitsu Limited | Dna computer and calculation method using the same |
WO2019246434A1 (en) * | 2018-06-20 | 2019-12-26 | Brown University | Methods of chemical computation |
US11989619B2 (en) | 2019-08-27 | 2024-05-21 | President And Fellows Of Harvard College | Modifying messages stored in mixtures of molecules using thin-layer chromatography |
-
1996
- 1996-08-21 WO PCT/US1996/013532 patent/WO1997007440A1/en active Application Filing
- 1996-08-21 JP JP50957197A patent/JP2001515614A/en active Pending
- 1996-08-21 AU AU67801/96A patent/AU6780196A/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
SCIENCE, 28 April 1995, Vol. 268, BAUM, E.B., "Building an Associative Memory Vastly Larger than the Brain", pages 583-585. * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001059704A1 (en) * | 2000-02-11 | 2001-08-16 | Universiteit Leiden | A biological molecule based computing method based on a blocking principle |
EP1124198A1 (en) * | 2000-02-11 | 2001-08-16 | Universiteit Leiden | A biological molecule based computing method based on a blocking principle |
EP1154376A1 (en) * | 2000-05-12 | 2001-11-14 | Universiteit Leiden | The use of proteinaceous molecules in methods for molecular computing |
WO2001086590A1 (en) * | 2000-05-12 | 2001-11-15 | Universiteit Leiden | The use of proteinaceous molecules in methods for molecular computing |
WO2004068398A1 (en) * | 2003-01-30 | 2004-08-12 | Fujitsu Limited | Dna computer and calculation method using the same |
US7167847B2 (en) | 2003-01-30 | 2007-01-23 | Fujitsu Limited | DNA computer and a computation method using the same |
WO2019246434A1 (en) * | 2018-06-20 | 2019-12-26 | Brown University | Methods of chemical computation |
US11093865B2 (en) | 2018-06-20 | 2021-08-17 | Brown University | Methods of chemical computation |
US11790280B2 (en) | 2018-06-20 | 2023-10-17 | Brown University | Methods of chemical computation |
US11989619B2 (en) | 2019-08-27 | 2024-05-21 | President And Fellows Of Harvard College | Modifying messages stored in mixtures of molecules using thin-layer chromatography |
Also Published As
Publication number | Publication date |
---|---|
JP2001515614A (en) | 2001-09-18 |
AU6780196A (en) | 1997-03-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Roweis et al. | A sticker based model for DNA computation. | |
Kok et al. | Natural Computing Series | |
Adleman | On constructing a molecular computer. | |
Paun et al. | DNA computing: new computing paradigms | |
Amos | DNA computation | |
Kari et al. | DNA Computing-Foundations and Implications. | |
US5804373A (en) | Molecular automata utilizing single- or double-strand oligonucleotides | |
El-Seoud et al. | DNA Computing: Challenges and Application. | |
EP1215623A2 (en) | Novel computation with nucleic acid molecules, computer and software for computing | |
Wang et al. | Parallel molecular computation on digital data stored in DNA | |
WO1997007440A1 (en) | Molecular computer | |
WO1997007440A9 (en) | Molecular computer | |
Gearheart et al. | DNA-based random number generation in security circuitry | |
GB2568974A (en) | Error detection during hybridisation of target double-stranded nucleic acid | |
Mills Jr et al. | Experimental aspects of DNA neural network computation | |
Kari | From Micro-Soft to Bio-Soft: Computing With DNA. | |
Amos | DNA Computing. | |
Pisanti | DNA computing: a survey | |
Sharma et al. | DNA computing: methodologies and challenges | |
Kari | DNA computing in vitro and in vivo | |
Gibbons et al. | Models of DNA computation | |
Rajaee et al. | A new approach for Boolean matrix multiplication with DNA computing | |
Ibrahim et al. | Solving unconstraint assignment problem by a molecular-based computing algorithm | |
Nowzari-Dalini et al. | A New DNA Implementation of Finite State Machines. | |
Krishna et al. | Breaking the Boundaries using DNA Technologies to Advance Computing |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
COP | Corrected version of pamphlet |
Free format text: PAGES 1/7-3/7 AND 5/7, DRAWINGS, REPLACED BY NEW PAGES 1/5 AND 3/5; PAGES 4/7,6/7 AND 7/7 RENUMBERED AS PAGES 2//5,4/5 AND 5/5; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 1997 509571 Kind code of ref document: A Format of ref document f/p: F |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: CA |