The present invention provides methods of identifying a plant containing a lesion in a gene sequence flanked in a wild type chromosome by known polynucleotide sequences. The methods comprise providing a collection of nucleic acids from source plants, providing a data base that associates each nucleic acid with its source plant, amplifying the collection of nucleic acids, thereby detecting the lesion, and using the database to identify the source plant carrying the lesion.

Not applicable.
In recent years, genomic sequencing of several organisms has been completed and genomic sequencing of many other organisms has also been initiated. Furthermore, the complete genomic sequences of many organisms, including human, some model organisms such as C. elegans, Drosophila and Arabidopsis, as well as some economically important crops and animals will be known within the next decade. Many of the genes identified from this research, however, will have unknown functions.
One approach to study gene functions is to knock out a gene of an organism and find out what happens to the mutant organism. Studying the mutant organism often leads to better understanding of the functions of the gene. Also, gene knockouts allow for the specifically elimination or change of undesirable traits.
Currently there are technologies available to create knock-out animals through homologous recombination. See, e.g., Galli-Taliadoros, et al. J. Immunol. Methods 181(1):1-15 (1995); Buerstedde, et al., Cell, 67:179-88 (1991); Jasin, et al., Genes and Devel., 2:1353-63 (1988); Sedivy, et al., Proc. Natl. Acad. Sci. USA, 86:227-31 (1989). Such technologies are expensive and inefficient. For unknown reasons, however, the frequency of homologous recombination in plants is so low that it is not possible to create a knock-out plant through homologous recombination. Although some researchers have succeeded in lowering expression levels of certain plant genes by making transgenic plants expressing antisense RNA of those genes, they can not completely eliminate the expression and function of those genes. In many cases, complete elimination of the function of a gene is needed. Also, it is currently either very difficult or impossible to make transgenic plants in many crop species.
Bensen et al. Plant Cell 7(1): 75-84 (1995) teach a method of identifying transposon insertion mutants in known maize gene sequences by amplifying the region between a transposon sequence and a particular gene sequence. This method, however, is limited to the few plant systems with characterized transposon systems or requires the introduction of such systems in heterologous plants. This method therefore does not provide a universal method for identification of mutations in known sequences of any plant species.
Therefore a need exists for obtaining and detecting a genetic lesion in a known plant gene sequence. Ideally, this method should be useful in as broad a range of plants as possible and therefore should not depend on such criteria as availability of transformation methods.
The present invention provides a method for identifying a plant containing a lesion in a gene sequence. The method comprises providing a collection or pool of nucleic acid samples wherein each sample is collected from an individual plant. Typically the individual plants used in the method are derived from a mutagenized population of source plants, thereby insuring genetic diversity within the population.
The method of the invention also provides a database that associates each nucleic acid sample with the source plant from which the nucleic acid sample was derived.
In addition, the method of the invention includes amplifying the collection of nucleic acids using primers that specifically hybridize to known polynucleotide sequences that flank the gene sequence in the wild type chromosome. In one embodiment, the amplification includes an extension step that is shortened to selectively amplify a nucleic acid with the lesion. For instance, the extension time can be between 0.5 and two minutes at 72xc2x0 C. In one embodiment, the extension time is 1.5 minutes.
After the nucleic acids are amplified, a genetic lesion within the gene sequence of one of the samples is detected. For instance, the lesion can be detected as a change in size of the amplification product. Once the nucleic acid sample containing the lesion is identified, the database is used to identify the source plant from which the nucleic acid sample was derived.
In one embodiment, the lesion is a deletion.
In another embodiment, the collection of nucleic acids is divided into subset pools before the lesion is detected. For example, the pools can contain nucleic acids from about 100 different to about 3000 different source plants. In another embodiment, the pools can contain at least about 1500 different source plants.
The plurality of source plants or the progenitors of the source plants can, for example, be contacted with a mutagen. In one embodiment, the mutagen is fast neutrons.
In one embodiment, the distance between the known nucleotide sequences in the wild type plant is between about three kilobases and seven kilobases.
The method of the invention can be performed, for instance, on a plurality of source plants such as rice, tomato or Arabidopsis. In one embodiment, the number of source plants is about 10,000 to about 50,000.
Included in the invention is a plant identified by the method described above.
In a preferred embodiment of the invention, a source plant carrying a deletion in a gene sequence is identified. In this embodiment, a collection of nucleic acids from a plurality of mutagenized plants, at least one of which carries a deletion in the gene sequence, is provided. A database is also provided that associates each nucleic acid in the collection with the nucleic acid""s source plant. The collection of nucleic acids is then amplified. The amplification comprises providing primers that specifically hybridize to polynucleotide sequences from about 3 kb to about 7 kb apart in the wild type chromosome and that specifically hybridize to polynucleotide sequences within or flanking the gene sequences. The amplification further comprises an extension step of between about 0.5 minutes to about two minutes at 72xc2x0 C., thereby preferentially amplifying the nucleic acids with the deletion. The presence of the nucleic acid with the deletion is confirmed by comparing the amplification product with an amplification product from a wild type plant. The database is then used to identify the source plant with the deletion, thereby providing a plant with a deletion in the gene sequence for evaluation of a mutant phenotype associated with the deletion.
Definitions
A xe2x80x9cgene sequencexe2x80x9d refers to a nucleic acid sequence that comprises a sequence or subsequence from an open reading frame and/or regulatory sequences in genomic DNA or a cDNA.
A xe2x80x9cwild type chromosomexe2x80x9d refers to a polynucleotide sequence that exists in a plant before human-initiated mutagenesis is performed. xe2x80x9cHuman initiated mutagenesisxe2x80x9d does not include, for instance, selection of agronomically valuable traits through selective breeding, but does include the introduction of mutagenic compounds or elements to promote genetic diversity in a population. Such mutagenic compounds include, e.g., transposons.
A xe2x80x9csource plantxe2x80x9d is a particular plant from which nucleic acids were harvested or the plant""s genetic equivalent. For the purposes of this invention, genetically equivalent plants are those plants from which the same specific genetic lesion can be recovered. Genetically equivalent plants includes siblings or progeny from a plant of interest. Thus, if M1 seeds are mutagenized, grown into M1 plants and their M2 seed are harvested, pooled M2 seeds from the same parent are the same xe2x80x9csource plantxe2x80x9d because a specific mutation of one M2 plant that was received from an M1 parent is likely to be found in a pool of M2 siblings from the same M1 parent. Similarly, if an M2 plant has a particular genetic lesion, pools of M3 progeny from the M2 plant will also likely carry that lesion.
A xe2x80x9clesionxe2x80x9d can be any molecular alteration of a nucleic acid relative to wild type plant nucleic acids. For instance, a lesion can be a deletion, inversion, insertion, duplication or a rearrangement in a nucleic acid sequence. The lesion is preferably deletion.
xe2x80x9cProgenitors of the plurality of source plantsxe2x80x9d are the ancestors of a particular collection of source plants. For instance, if M1 seed is mutagenized, grown into M1 plants and then M2 seed is collected from the M1 plants, the M1 plants are progenitors of the M2 plants.
xe2x80x9cEvaluation of a mutant phenotypexe2x80x9d refers to identifying physical traits of a plant carrying a genetic lesion that differ from the traits of a wild type plant. The manifestation of such traits can be dependent on the genetic makeup of the plant, e.g. whether the plant is homozygous or heterozygous for a particular allele, including the allele containing the lesion, as well as the overall genetic background. If the lesion causes a homozygous lethal phenotype then the lesion can be maintained in future generations in the heterozygous state
The phrase xe2x80x9cnucleic acid sequencexe2x80x9d refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5xe2x80x2 to the 3xe2x80x2 end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
The term xe2x80x9cpromoterxe2x80x9d refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9cplant promoterxe2x80x9d is a promoter capable of initiating transcription in plant cells.
The term xe2x80x9cplantxe2x80x9d includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
A polynucleotide sequence is xe2x80x9cheterologous toxe2x80x9d an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).
xe2x80x9cRecombinantxe2x80x9d refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley and Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.
A polynucleotide xe2x80x9cexogenous toxe2x80x9d an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T1 (e.g. in Arabidopsis by vacuum infiltration) or R0 (for plants regenerated from transformed cells in vitro) generation transgenic plant. Transgenic plants that arise from sexual cross or by selfing are descendants of such a plant.
Two nucleic acid sequences or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
One of skill in the art will recognize that two polypeptides can also be xe2x80x9csubstantially identicalxe2x80x9d if the two polypeptides are immunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides display significant variation. Therefore a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A xe2x80x9ccomparison windowxe2x80x9d, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins and Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra) These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat""l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g., Creighton, Proteins (1984)).
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
The phrase xe2x80x9cselectively (or specifically) hybridizes toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Probes, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid assaysxe2x80x9d (1993). Generally, highly stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30xc2x0 C. below the Tm. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
In the present invention, genomic DNA or cDNA comprising ANT nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and at least one wash in 0.2xc3x97SSC at a temperature of at least about 50xc2x0 C., usually about 55xc2x0 C. to about 60xc2x0 C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary xe2x80x9cmoderately stringent hybridization conditionsxe2x80x9d include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and a wash in 1xc3x97SSC at 45xc2x0 C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., an RNA gel or DNA gel blot hybridization analysis.
The present invention provides, for the first time, a method for determining the function of a gene sequence by screening for a genetic lesion in the gene sequence. The method involves identifying plants carrying the genetic lesion by amplifying a polynucleotide sequence comprising the gene sequence from a large number of plants and then comparing the amplification products with an amplification product from wild type plants. In preferred embodiments, a nucleic acids from a plurality of source plants are pooled and amplified. In a preferred embodiment, amplification conditions are provided such that nucleic acids with a genetic lesion in a particular gene sequence is preferentially amplified.
Plant Sources of the Invention
The present invention can be used with any species of plant. The invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoseyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea. In preferred embodiments, Oryza (rice), Lycopersicon (tomato) or Arabidopsis are used.
The present invention provides a collection of nucleic acids from a plurality of plants. The plurality of plants can be any collection of plants that carry genetic variation. In a preferred embodiment of the invention, the plurality of plants or the progenitors of the plants have been exposed to a mutagen likely to cause genetic lesions, thereby promoting genetic variation within the population. The mutagen is preferably chosen so as to produce a high percentage of deletions.
Plant mutatgenesis
A mutagenized population of seeds can be obtained or created. For instance, an existing mutagenized population of plants can be ordered or obtained from commercial or non-commercial suppliers (see, e.g., Lehle Seed, Round Rock, Texas and the Arabidopsis Biological Resource Center for mutagenized Arabidopsis seeds). Alternatively, a mutagenized population can be developed for any plant species. For instance, seeds or other plant material can be treated with a mutagenic chemical substance, according to standard techniques. Such chemical substances include, but are not limited to, the following: diepoxybutane, diethyl sulfate, ethylene imine, ethyl methanesulfonate and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from sources such as, for example, X-rays, gamma rays or fast neutron bombardment can be used.
A mutagenized population is typically developed by generating a large number of seed (xcx9c50,000) and exposing the seed to a mutagen. One of skill will recognize that a series of concentrations and time of exposures is used to determine the proper level of mutagenesis. Typically such test populations are planted in soil or plated onto solid growth media. The number of seed that do not germinate is a good indicator of the extent of mutagenesis. Preferably the mutagenized population will typically have a high enough frequency of mutation to identify the desired mutation in as small a number of plants possible, while avoiding over-mutagenizing the population such that a typical plant carries multiple different genetic lesions and to ensure that most plants grown from the mutagenized seeds will produce seeds for the next generation. Theoretically, an ideal dose will be the one that makes about 37% of plants grown from the treated seeds sterile.
In one embodiment, seed (designated M1) are mutagenized, grown into plants, and M2 seed is harvested from them. Plant populations with plants that are homozygous and heterozygous for genetic lesions (e.g., M2 populations) are preferably used as the nucleic acid source for the present invention. Typically M2 plants are grown and leaf or root tissue is harvested for nucleic acid isolation. M3 seed can be collected from the M2 plant, thereby producing a source plant population that can be later used to recover any genetic lesion in the parent M2 plant. Alternatively, the siblings (as seed or plants) of the M2 plant are saved to provide a source plant population. This second embodiment saves plant growing space but adds an additional step later to recover a desired genetic lesion.
Type of genetic lesions
Mutagens can produce, inter alia, point mutations, deletions, inversions, insertions, duplications and rearrangements. Genetic lesions can be large or small. For the present invention, a preferred lesion significantly changes the distance between the primers that hybridize to the two known polynucleotide sequences. Such lesions include deletions, insertions and duplications. A significant change is one that can be readily detected using standard procedures for specifically detecting nucleic acids, e.g., PCR. Typically a change of greater than at least 30% of the distance between the primers that hybridize to the two known polynucleotides is preferred. In an especially preferred embodiment, the genetic lesion is a deletion that removes from at least about 10% to about 85% of the distance between the primers. For instance, in a typical case, the primers are selected to be about 7 kb apart and the deletion is about 0.5 kb to about 6 kb.
Preparation of nucleic acids from the plurality of plants
Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer""s specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley and Sons, Inc. (1994-1998).
Databases of the Invention
In preferred embodiments of the present invention, nucleic acids are isolated from individual plants and then subsequently pooled to perform the method of the invention. A unique number or code is provided for nucleic acid preparations from each individual plant and the same name or code is used to track and maintain each source plant population.
In one embodiment, the assays of this invention are facilitated by the use of databases to record assay results. Particularly with the use of large-scale screening systems, (e.g., screening large populations of planta )data management can be extremely useful. Maintenance and management of the information obtained by screening such a library is aided by methods automated information retrieval, e.g. a computer database.
Such a database is useful for a variety of functions, including, but not limited to library registration, library or result display, library and/or result specification, documentation, and data retrieval and exploratory data analysis. The registration function of a database provides recordation/registration of plant and nucleic acid sources. Library and screning result display functions provide an effective means to review and/or categorize relevant assay data. The database also can provide documentation of screening results and the ability to rapidly retrieve, correlate (or statistical analysis), and evaluate assay data.
The database can be any medium convenient for recording and retrieving information generated by the assays of this invention. Such databases include, but are not limited to manual recordation and indexing systems (e.g. file-card indexing systems). However, the databases are most useful when the data therein can be easily and rapidly retrieved and manipulated (e.g. sorted, classified, analyzed, and/or otherwise organized). Thus, in a preferred embodiment, the signature the databases of this invention are most preferably xe2x80x9cautomatedxe2x80x9d, e.g., electronic (e.g. computer-based) databases. The database can be present on an individual xe2x80x9cstand-alonexe2x80x9d computer system, or a component of or distributed across multiple xe2x80x9cnodesxe2x80x9d (processors) on a distributed computer systems. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to xe2x80x9cpersonal computer systemsxe2x80x9d, mainframe systems, distributed nodes on an inter- or intra-net, data or databases stored in specialized hardware (e.g. in microchips), and the like.
Amplification
The invention provides for an amplification step to generate an appropriate amount of nucleic acids to resolve difference in a particular sequence between a nucleic acid carrying a lesion and a wild type nucleic acid.
PCR
In a preferred embodiment, the polymerase chain reaction (PCR) is used to amplify both wild type and mutant DNA within a pooled sample. PCR, as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al., describe a method for increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification. PCR can be used to directly increase the concentration of the target to an easily detectable level. The process for amplifying the target sequence involves introducing a molar excess of two oligonucleotide primers that are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize. Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization, and polymerase extension can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990).
The length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, as well as the length of time that the polymerase is allowed to extend the DNA strand (xe2x80x9cthe extension timexe2x80x9d). Therefore, this length is a controllable parameter.
In a preferred embodiment of the present invention, an extension time is chosen such that nucleic acids containing the genetic lesion are preferentially amplified. For instance, if the genetic lesion is suspected to be a deletion, a shorter extension time is used so that proper amplification only occurs if the distance between the primers is less than is found in a wild type plant. One of skill can design amplification conditions based on the expected deletion size, distance between the primers, and the like. For example, in some embodiments, the extension time can be from about one minute to about two minutes at 72xc2x0 C. In a more preferred embodiment, the extension time is 1.5 minutes at 72xc2x0 C.
Designing primers
Primers appropriate for the invention will depend upon the gene sequence of interest. Primer construction and use are well known to those of skill in the art. In preferred embodiments, the primers are constructed to hybridize to polynucleotides that flank the gene sequence. The polynucleotide sequences can flank the coding region of the gene of interest or they can flank the coding sequence and regulatory sequences (e.g., promoter and 3xe2x80x2 regions of the gene). Alternatively, the primers can be constructed to hybridize within the regulatory or coding regions of the gene. It is preferred that the primers hybridize to polynucleotides from about 3 kb to about 7 kb apart.
In species where extensive DNA sequencing has been performed, such as Arabidopsis, it is likely that genomic sequences flanking a gene sequence of interest will be known. If so, primer sequences can be developed from existing known genomic sequences. If the genomic sequences flanking a gene sequence of interest are not available, known polynucleotide sequences that flank the gene sequence of interest can be developed. For instance, genomic libraries (cosmid, BAC, YAC, etc.) can be screened for clones that contain the gene of interest and the ends of the inserts in such clones can be sequenced. Alternatively, restriction enzyme mapping of a particular genomic region can be performed, followed by nucleotide sequencing of the region flanking the gene of interest. Primers can then be derived from such sequences.
Long Range PCR
In one embodiment of the invention, the collection of nucleic acids are amplified using long range PCR (see, e.g., 5. Page 17, line 13, Proc. Natl. Acad. Sci. U.S.A. 9: 2216-2220, (1994); Blood, 88(3): 985-994 (1996)) Recently several thermostable DNA polymerases that can amplify DNA fragments up to 35 kb have become available commercially. By designing primer pairs separated by a long distance of up to 35 kb, a significantly larger region can be amplified.
To analyze such large nucleic acids, the amplified DNA fragment is then digested by a restriction enzyme and analyzed by gel electrophoresis. The DNA can be detected by staining the gel with ethidium bromide or fluorescent dye. Preferably, DNA deletions or rearrangements within the region will create additional DNA fragment(s) compared to wild type. Long range PCR is also useful to detect large deletion around a gene.
Other amplification methods
The desired nucleic acids can also be identified using other well known amplification techniques. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the ligase chain reaction (LCR), Qxcex2-replicase amplification and other RNA polymerase mediated techniques are found in Berger, Sambrook, and Ausubel, as well as Arnheim and Levinson (Oct. 1, 1990) CandEN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem. 35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117. Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.
Analysis of amplification products
Analysis of the amplification products comprises comparing the amplification products from a sample (e.g. a plant containing a genetic lesion or products from a pool of plants, one or more of which contain the lesion) to a similar amplification product of a wild type plant. Preferably the amplification products are separated based on their length. Alterations in the size of the sample amplification product compared to the wild type product indicates that a nucleic acid with a genetic lesion at the gene sequence of interest is in the sample. For instance, amplification of a smaller DNA fragment from the sample than from the wild type indicates that at least one individual plant from the sample has a deletion within the gene sequence of interest. This result can be confirmed and if the sample is a pool of nucleic acids, then subpools or individual members of the pool can be individually amplified to identify the nucleic acid carrying the lesion. Once the nucleic acid is identified, the database can be used to identify the source plant associated with the nucleic acid.
The amplification products can be analyzed by a number of techniques, including electrophoresis and hybridization. Preferably the amplification products are analyzed using electrophoresis and ethidium bromide staining (see, e.g., Sambrook, et al.), followed by visual inspection.
Alternatively, to increase the detection limit of the amount of DNA fragments on the gel, the DNA fragments can be transferred to a membrane by southern blotting and hybridized with radioactive or nonradioactive probes specific to the target gene. After hybridization, very small amount of DNA ( less than 1 pg) can be detected by autoradiography or other detection methods. Because of this great sensitivity, a large number of DNA samples can be pooled together as one sample and checked for deletion mutation(s). This embodiment is particularly helpful if it is difficult to resolve the amplification products clearly using ethidium bromide followed by visual inspection.
Hybridization
An alternative method of resolving differences between wild type and pools carrying mutant alleles of a gene involves hybridization. In this embodiment, pools of nucleic acids from mutagenized plants are digested with restriction enzymes and the resulting nucleic acid fragments are separated by electrophoresis and blotted (e.g. Southern blotting). See, e.g., Sambrook et al. The resulting blots are subsequently probed with a probe of the gene of interest and compared to similar samples of wild type nucleic acids. Hybridization of the probe in the pooled sample to nucleic acid fragments of different size than wild type indicates a genetic lesion exists in the pool. The method can then be repeated with individual members of the pool to determine which individual carries the lesion. As described above, once the nucleic acid is identified, the database can be used to identify the source plant associated with the nucleic acid.
Determination of a Mutant Phenotype
The particular endogenous sequence targeted in the methods of the invention is not a critical aspect of the invention. Examples of genes that can be targeted using the present invention include genes conferring resistance to pathogens (for example, insects, fungi, bacteria and viruses), storage protein genes, herbicide resistance genes, and genes involved in biosynthetic pathways, such as oil production. Other examples include Cytochrome P450 and related enzymes, transcription factors, genes that are essential for plant growth and development, and homologs of genes with known function.
Plants identified by the methods of the invention (e.g., mutants) may have useful phenotypes themselves. For instance, mutations in certain biosynthetic pathways can lead to a useful alteration of a plant""s metabolism. Alternatively, once the phenotype of a mutant is known, ectopic or heterologous expression of the wild type gene can be hypothesized and tested. In particular, appropriate genetic backgrounds can be identified to alter plant phenotypes. For example, the phenotypic expression of a particular transgene may be concealed by a wild type homolog or ortholog. Once a mutant allele""s phenotype is determined, it is possible to identify genetic backgrounds where such effect is limited.

Method for obtaining a plant with a genetic lesion in a gene sequence


A hand-held washing device comprises a scrubber and a fluid dispenser to contain a cleaning fluid such as liquid soap. The device is constructed of an mesh material bound together in a plurality of pleats. The fluid dispenser includes a fill opening and an outlet. A plug is sized to be removable mounted within the fill opening. When mounted, the plug prevents liquid within the fluid dispenser from escaping through the fill opening.

The present invention is directed to a hand-held washing device and, more particularly, a washing device constructed of elongated material folded into a plurality of pleats containing a fluid dispenser containing liquid soap and the like.
A variety of hand-held washing devices are available such as scrub brushes, sponges, and the like. These require that soap be separately applied prior to using the device. After the soap is applied, the device is used for scrubbing or otherwise distributing the soap along a user's body. This is inconvenient as it either requires the user to hold the soap in one hand and the device in the other, or requires the user to repeatedly pick up and set down the device and soap as needed.
Some designs feature integrated soap contained within the device. However, these designs are difficult for the user as the soap either cannot be refilled or is difficult to replenish once depleted. Designs that cannot be refilled are obviously not effective after they are extinguished and must be replaced by a new device. Designs that are difficult to refill may not be favored by users. Refilling of these devices is often difficult because the soap is located in the device interior that is difficult to access. Conversely, some designs feature the soap on the device exterior which is easy to access, but may result in the soap repeatedly falling out of the device, or scratching the user during use such as when the soap is contained within a fluid dispenser.
The device should further be designed such that the soap is not damaged or discharged when not in use. By way of example, the bar of soap stored within a wet device will become soft and fall apart during a subsequent washing. Fluid dispensers should be able to contain the washing liquid such that it does not leak or discharge when the device is not in use.
It is also advantageous that the device be aesthetically pleasing. Washrooms and bathrooms are often fancifully decorated and users do not want to put an unattractive object within the environment. It is advantageous if the device itself can further add to the aesthetic qualities of the room.


The connection architecture of a network on a chip (NoC) is described in which (a) nodes in octahedron sections are connected in an arc Benes network, (b) a hierarchy of node clusters are connected using a globally asynchronous locally asynchronous (GALA) configuration, (c) a double wishbone 2D torus ring is applied to connection between network layers and (d) data is routed using buffer modulation.

The challenge of modern computing is to build economically efficient chips that incorporate more transistors to meet the goal of achieving Moore's law of doubling performance every two years. The limits of semiconductor technology are affecting this ability to grow in the next few years, as transistors become smaller and chips become bigger and hotter. The semiconductor industry has developed the system on a chip (SoC) as a way to continue high performance chip evolution.
So far, there have been four main ways to construct a high performance semiconductor. First, chips have multiple cores. Second, chips optimize software scheduling. Third, chips utilize efficient memory management. Fourth, chips employ polymorphic computing. To some degree, all of these models evolve from the Von Neumann computer architecture developed after WWII in which a microprocessor's logic component fetches instructions from memory.
The simplest model for increasing chip performance employs multiple processing cores. By multiplying the number of cores by eighty, Intel has created a prototype teraflop chip design. In essence, this architecture uses a parallel computing approach similar to supercomputing parallel computing models. Like some supercomputing applications, this approach is limited to optimizing arithmetic-intensive applications such as modeling.
The Tera-op, Reliable, Intelligently Adaptive Processing System (TRIPS), developed at the University of Texas with funding from DARPA, focuses on software scheduling optimization to produce high performance computing. This model's "push" system uses data availability to fetch instructions, thereby putting additional pressure on the compiler to organize the parallelism in the high speed operating system. There are three levels of concurrency in the TRIPS architecture, including instruction-level parallelism (ILP), thread-level parallelism (TLP) and data-level parallelism (DLP). The TRIPS processor will process numerous instructions simultaneously and map them onto a grid for execution in specific nodes. The grid of execution nodes is reconfigurable to optimize specific applications. Unlike the multi-core model, TRIPS is a uniprocessor model, yet it includes numerous components for parallelization.
The third model is represented by the Cell microprocessor architecture developed jointly by the Sony, Toshiba and IBM (STI) consortium. The Cell architecture uses a novel memory "coherence" architecture in which latency is overcome with a bandwidth priority and in which power usage is balanced with peak computational usage. This model integrates a microprocessor design with coprocessor elements; these eight elements are called "synergistic processor elements" (SPEs). The Cell uses an interconnection bus with four unidirectional data flow rings to connect each of four processors with their SPEs, thereby meeting a teraflop performance objective. Each SPE is capable of producing 32 GFLOPS of power in the 65 nm version, which was introduced in 2007.
The MOrphable Networked Micro-ARCHitecture (MONARCH) uses six reduced instruction set computing (RISC) microprocessors, twelve arithmetic clusters and thirty-one memory clusters to achieve a 64 GFLOPS performance with 60 gigabytes per second of memory. Designed by Raytheon and USC/ISI from DARPA funding, the MONARCH differs distinctly from other high performance SoCs in that it uses evolvable hardware (EHW) components such as field programmable compute array (FPCA) and smart memory architectures to produce an efficient polymorphic computing platform.
MONARCH combines key elements in the high performance processing system (HPPS) with Data Intensive Architecture (DIVA) Processor in Memory (PIM) technologies to create a unified, flexible, very large scale integrated (VLSI) system. The advantage of this model is that reprogrammability of hardware from one application-specific integrated circuit (ASIC) position to another produces faster response to uncertain changes in the environment. The chip is optimized to be flexible to changing conditions and to maximize power efficiency (3-6 GFLOPS per watt). Specific applications of MONARCH involve embedded computing, such as sensor networks.
These four main high performance SoC models have specific applications for which they are suited. For instance, the multi-core model is optimized for arithmetic applications, while MONARCH is optimized for sensor data analysis. However, all four also have limits.
The multi-core architecture has a problem of synchronization of the parallel micro-processors that conform to a single clocking model. This problem limits their responsiveness to specific types of applications, particularly those that require rapid environmental change. Further, the multi-core architecture requires "thread-aware" software to exploit its parallelism, which is cumbersome and produces quality of service (QoS) problems and inefficiencies.
By emphasizing its compiler, the TRIPS architecture has the problem of optimizing the coordination of scheduling. This bottleneck prevents peak performance over a prolonged period.
The Cell architecture requires constant optimization of its memory management system, which leads to QoS problems.
Finally, MONARCH depends on static intellectual property (IP) cores that are limited to combinations of specified pre-determined ASICs to program its evolvable hardware components. This restriction limits the extent of its flexibility, which was precisely its chief design advantage.
In addition to SoC models, there is a network on a chip (NoC) model, introduced by Arteris in 2007. Targeted to the communications industry, the 45 nm NoC is a form of SoC that uses IP cores in FPGAs for reprogrammable functions and that features low power consumption for embedded computing applications. The chip is optimized for on-chip communications processing. Though targeted at the communications industry, particularly wireless communications, the chip has limits of flexibility that it was designed to overcome, primarily in its deterministic IP core application software.
Various implementations of FPGAs represent reconfigurable computing. The most prominent examples are the Xilinx Virtex-II Pro and Virtex-4 devices that combine one or more microprocessor cores in an FPGA logic fabric. Similarly, the Atmel FPSLIC processor combines an AVR processor with programmable logic architecture. The Atmel microcontroller has the FPGA fabric on the same die to produce a fine-grained reconfigurable device. These hybrid FPGAs and embedded microprocessors represent a generation of system on a programmable chip (SOPC). While these hybrids are architecturally interesting, they possess the limits of each type of design paradigm, with restricted microprocessor performance and restricted deterministic IP core application software. Though they have higher performance than a typical single core microprocessor, they are less flexible than a pure FPGA model.
All of these chip types are two dimensional planar micro system devices. A new generation of three dimensional integrated circuits and components is emerging that is noteworthy as well. The idea to stack two dimensional chips by sandwiching two or more ICs using a fabrication process required a solution to the problem of creating vertical connections between the layers. IBM solved this problem by developing "through silicon vias" (TSVs) which are vertical connections "etched through the silicon wafer and filled with metal." This approach of using TSVs to create 3D connections allows the addition of many more pathways between 2D layers. However, this 3D chip approach of stacking existing 2D planar IC layers is generally limited to three or four layers. While TSVs substantially limit the distance that information traverses, this stacking approach merely evolves the 2D approach to create a static 3D model.
In U.S. Pat. No. 5,111,278, Echelberger describes a 3D multi-chip module system in which layers in an integrated circuit are stacked by using aligned TSVs. This early 3D circuit model represents a simple stacking approach. U.S. Pat. No. 5,426,072 provides a method to manufacture a 3D IC from stacked silicon on insulation (SOI) wafers. U.S. Pat. No. 5,657,537 presents a method of stacking two dimensional circuit modules and U.S. Pat. No. 6,355,501 describes a 3D IC stacking assembly technique.
Recently, 3D stacking models have been developed on chip in which several layers are constructed on a single complementary metal oxide semiconductor (CMOS) die. Some models have combined eight or nine contiguous layers in a single CMOS chip, though this model lacks integrated vertical planes. MIT's microsystems group has created 3D ICs that contain multiple layers and TSVs on a single chip.
3D FPGAs have been created at the University of Minnesota by stacking layers of single planar FPGAs. However, these chips have only adjacent layer connectivity.
3D memory has been developed by Samsung and by BeSang. The Samsung approach stacks eight 2-Gb wafer level processed stack packages (WSPs) using TSVs in order to minimize interconnects between layers and increase information access efficiency. The Samsung TSV method uses tiny lasers to create etching that is later filled in with copper. BeSang combines 3D package level stacking of memory with a logic layer of a chip device using metal bonding.
See also U.S. Pat. No. 5,915,167 for a description of a 3D DRAM stacking technique, U.S. Pat. No. 6,717,222 for a description of a 3D memory IC, U.S. Pat. No. 7,160,761 for a description of a vertically stacked field programmable nonvolatile memory and U.S. Pat. No. 6,501,111 for a description of a 3D programmable memory device.
Finally, in the supercomputing sphere, the Cray T3D developed a three dimensional supercomputer consisting of 2048 DEC Alpha chips in a torus networking configuration.
In general, all of the 3D chip models merely combine two or more 2D layers. They all represent a simple bonding of current technologies. While planar design chips are easier to make, they are not generally high performance.
Prior systems demonstrate performance limits, programmability limits, multi-functionality limits and logic and memory bottlenecks. There are typically trade-offs of performance and power.
The present invention views the system on a chip as an ecosystem consisting of significant intelligent components. The prior art for intelligence in computing consists of two main paradigms. On the one hand, the view of evolvable hardware (EHW) uses FPGAs as examples. On the other hand, software elements consist of intelligent software agents that exhibit collective behaviors. Both of these hardware and software aspects take inspiration from biological domains.
First, the intelligent SoC borrows from biological concepts of post-initialized reprogrammability that resembles a protein network that responds to its changing environmental conditions. The interoperation of protein networks in cells is a key behavioral paradigm for the SoC. The slowly evolving DNA root structure produces the protein network elements, yet the dynamics of the protein network are interactive with both itself and its environment.
Second, the elements of the SoC resemble the subsystems of a human body. The circulatory system represents the routers, the endocrine system is the memory, the skeletal system is comparable to the interconnects, the nervous system is the autonomic process, the immune system provides defense and security as it does in a body, the eyes and ears are the sensor network and the muscular system is the bandwidth. In this analogy, the brain is the central controller.
For the most part, SoCs require three dimensionality in order to achieve high performance objectives. In addition, SoCs require multiple cores that are reprogrammable so as to maintain flexibility for multiple applications. Such reprogrammability allows the chip to be implemented cost effectively. Reprogrammability, moreover, allows the chip to be updatable and future proof. In some versions, SoCs need to be power efficient for use in embedded mobile devices. Because they will be prominent in embedded devices, they also need to be fault tolerant. By combining the best aspects of deterministic microprocessor elements with indeterministic EHW elements, an intelligent SoC efficiently delivers superior performance.
While the design criteria are necessary, economic efficiency is also required. Computational economics reveals a comparative cost analysis that includes efficiency maximization of (a) power, (b) interconnect metrics, (c) transistor per memory metrics and (d) transistor per logic metrics.
Problems that the System Solves
Optimization problems that the system solves can be divided into two classes: bi-objective optimization problems (BOOPs) and multi-objective optimization problems (MOOPs).
BOOPs consist of trade-offs in semiconductor factors such as (a) energy consumption versus performance, (b) number of transistors versus heat dissipation, (c) interconnect area versus performance and (d) high performance versus low cost.
Regarding MOOPs, the multiple factors include: (a) thermal performance (energy/heat dissipation), (b) energy optimization (low power use), (c) timing performance (various metrics), (d) reconfiguration time (for FPGAs and CPLDs), (e) interconnect length optimization (for energy delay), (f) use of space, (g) bandwidth optimization and (h) cost (manufacture and usability) efficiency. The combination of solutions to trade-offs of multiple problems determines the design of specific semiconductors. The present system presents a set of solutions to these complex optimization problems.
One of the chief problems is to identify ways to limit latency. Latency represents a bottleneck in an integrated circuit when the wait to complete a task slows down the efficiency of the system. Examples of causes of latency include interconnect routing architectures, memory configuration and interface design. Limiting latency problems requires the development of methods for scheduling, anticipation, parallelization, pipeline efficiency and locality-priority processing.
Summary
The present invention features a network on a chip (NoC) in the form of a dynamic 3D intelligent system on a chip (iSoC). Chip network topology, routing architecture and flow dynamics are critical to the performance of the 3D iSoC. The 3D iSoC features novel networking features involving the structure and function of interconnects that markedly improve efficiency relative to other models.
The present invention describes a hybrid network that shares direct and indirect network architectures. In direct networks, each node is connected by a router to each other node, while in indirect networks, each node is connected to a switch which is then connected to other switches which connect to other nodes.
The network model used in the present invention is a hierarchical synthesis of direct and indirect networks. Each octagonal neighborhood has direct point-to-point connections via embedded routers between nodes, while each neighborhood has a switch that connects to both other neighborhoods and to the central core. This configuration benefits the independent operation of each neighborhood as well as the overall operation of the whole chip.
Analogous of the network configuration of the 3D iSoC is the structure and operation of a city. In the center core is a bigger set of larger buildings, while the periphery has multiple independent neighborhoods with smaller buildings. The city has different districts, such as warehouse district (memory), industrial district, shopping district, wholesale district and so on that perform specific functions. Overall, the city combines multiple different functions into a complex whole.
Further employing the analogy, the transportation system of the city is critical to the overall operation. Regarding the structure of the transportation system, a large highway will generally encircle a downtown center, with major arteries leading to the suburbs. Workers will travel from their homes to their offices in cars, buses and trains. The people are like individual data sets distributed in data packets of differentiated size (car versus a train). Organization, and reorganization, of traffic flows in the transportation subsystems determine the healthy functioning of the city. If a road is blocked, traffic backs up and a bottleneck is created. In this event, traffic is rerouted around the disruption. Once the roadway is cleared, traffic will resume its ordinary operations.
In the case of the 3D architecture of an intelligent SoC, multiple dimensions of symmetry extend the analogy. The chip's interconnects are the highways, yet in the 3D context they are even more analogous to the symmetrical functioning of the circulatory system of the body.
The present system introduces a hybrid 3D network in a SoC. The network consists of (a) an arc Benes network for rearrangable intra-neighborhood structure, (b) a 3D clos network with medium bandwidth to connect neighborhoods with hybrid synchronization, (c) globally asynchronous locally asynchronous (GALA) connections using crossbars to connect the central node to the neighborhoods (d) a multi-layer mesochronous communications matrix and (e) a double wishbone 2D torus model with highest bandwidth connecting the main quadrants.
The present disclosure describes solutions to problems involving interconnect structure and dynamics and routing constraints in the 3D environment of a ULSI circuit.
Novelties
The SoC is structured as a dynamic network in which clusters of connection nodes produce a variable configuration contingent on system demands. Continuous network optimization is performed by adaptive routing mechanisms. The hybrid networking system is also scalable.
Advantages of the Present System
The system uses efficient interconnect configurations for maximum energy conservation and energy leakage loss minimization. The increased number of symmetrically configured interconnections in a complex SoC with multiple multi-layer nodes also enables faster throughput, which leads to high performance.
The system makes possible polymorphous computing by employing a hybrid network control model that allows for a globally asynchronous locally asynchronous (GALA) hierarchical computing architecture. The clock speeds of the individual octahedron neighborhoods are variable, which leads to modulated, and efficient, power consumption.
Description of the Invention
(I) Intra-SoC Network Architecture
(1) Hybrid Network Fabric Integrating 3D Geodesic Interconnect Typology
The 3D NoC is fundamentally a network of interconnects distributed between logic and memory components. Interconnects embedded in the circuits link the logic circuitry and the memory circuitry to each layer, while the vias connect the circuitry of one layer of a 3D chip to other layers. Interconnects between chips provide the communications capacity for the SoC to operate as a network with a common switching fabric. Because the SoC is three dimensional, it uses a geodesic configuration to connect the various 3D nodes in Euclidean space using x, y and z dimensions.
Like a cube, the 3D aspect of the chip has six facades with eight corners. The eight corners correspond to a neighborhood cluster of circuit nodes, though the composition of each neighborhood cluster is variable. This configuration into neighborhood clusters allows each cluster to behave autonomously while also interacting with other clusters. The cluster configuration allows multiple nodes to operate independently and in parallel with other nodes.
The interconnects that link the nodes in each neighborhood are structured as a 3D geodesic architecture, with xy, yz and xz (top-down, right-left and front-back) directionality in each neighborhood cluster. These interconnects are like multi-lane roads with two-way traffic. The advantage of using two-way interconnects is to maintain efficiency; employing two one-way connections is not an efficient use of space.
In another embodiment of the invention, each node contains an RF wireless transmitter and receiver for broadcasting data to and receiving data from other nodes. Each node uses a separate bandwidth frequency for identification.
(2) 3D Torus Interconnect and Via Network for Layer to Layer Intranode Connection in 3D NoC and Method for Routing Therein
The use of through silicon vias (TSVs) in a 3D circuit allows the layers of each node to be connected. The present invention uses multiple TSV connections between each adjacent layer and multiple TSVs between non-adjacent layers. In particular, TSVs connect tiles of circuitry on a specific layer to tiles on other layers in the multi-layer integrated circuit. One way to organize this model is to use a planar controller on the side of the multi-layer chip that has access to each layer, much like a bank of elevator shafts.
The present system uses a 3D torus interconnect and via network to connect different layers within a multi-layer circuit and to route information from point to point. This model links the top and bottom layers by using an intermediary layer in the middle. The system's use of intra-layer TSVs maintains extreme efficiency.
In one configuration, alternating memory layers are sandwiched between logic layers. The memory layers have controllers that manage the memory features plus a routing mechanism that routes data to top and bottom layers.
The present system uses a TSV model of inverted broad-based pyramid structures within specific layers. The pyramid structures etch the TSVs in a configuration to connect tiles of specific layers to tiles of adjacent layers. This pattern is reproduced to connect tiles of multiple layers beyond the adjacent layers.
In this model, the interconnects and TSVs connecting the layers in the center of the 3D circuit are more used than peripheral layers. Consequently, the interconnects and TSVs in the central layers have a higher bandwidth than the interconnects and TSVs on the periphery of the chip. Their central location is more strategic and will require increased throughput capability.
(3) Arc Benes Network for Rearrangable Inter-node Connection in 3D NoC
There are eight neighborhood clusters in an NoC organized in a 3D arc Benes network configuration. The 3D Benes network is a form of fat tree communications architecture that connects nodes with vertical and horizontal interconnects in a geometrical configuration similar to the corner of a box. In the present system, the precise constitution of the set of nodes comprising a neighborhood cluster is variable. While the exterior node in each corner and the interior node in each corner are always included in the network cluster configuration, the addition of adjoining nodes will vary contingent upon a specific application. Generally, a neighborhood cluster configuration will have at least four nodes but may have as many as eight. This ad hoc, flexible and on-demand configuration of a neighborhood cluster provides maximum reprogrammability functioning of the overall NoC so as to optimize operations for various applications.
The 3D arc Benes network model uses the interior node and the exterior node of each SoC cubic configuration corner as shared router nodes. The peripheral nodes in the neighborhood will congregate and readjust into specific clusters around these two nodes.
The nodes in a neighborhood cluster have point to point connections. Since the constitution of neighborhood cluster configurations periodically vary, the point to point connections extend to the potential nodes in adjacent neighborhoods. The 3D arc Benes network represents a hybrid connection architecture that optimizes this point to point interconnection scheme between individual nodes as well as the connection architecture between the individual nodes in a neighborhood and the two corner nodes.
Because the interconnects are two-way, they route data in either direction between nodes simultaneously, thereby maximizing throughput capabilities.
The variable neighborhood configuration is critical in order to maintain adaptability and plasticity in 3D NoC reprogramming behaviors.
(4) 3D Clos 8-point Internode Interconnect Structure Using Globally Asynchronous Locally Asynchronous (GALA) Method for Hybrid Hierarchical Network in 3D NoC
While the neighborhood cluster architecture is useful to divide functions in a reprogrammable SoC, the challenge of connecting the octagonal clusters remains. The present system connects the eight neighborhood clusters organized into the corners of a cubic configuration by using a 3D clos network configuration. The inner nodes of each corner connect each neighborhood cluster to an interior connection matrix that connects both the central master core and the other neighborhood clusters.
Though connected to other parts of the 3D NoC network, each neighborhood cluster operates independently and uses its own adjustable clocking regulatory mechanism. This timing schema provides a locally asynchronous process in which each neighborhood cluster's node clocks are adjustable. This asynchronicity is necessary to accommodate the variable composition of each cluster. Once the set of nodes in each cluster is arranged for a particular application, the clocking for the nodes is harmonized for that application.
The linkage of clocking speeds with the other neighborhoods is also asynchronous because other neighborhoods are continuously modifying their clocking structures and thus their variable clocking synchronicity. The linkage of the neighborhoods with the central master core produces a clocking mechanism that is in perpetual disequilibrium.
The advantage of utilizing the GALA network architecture is the construction of a push-pull functional model. From the "top", data are pushed to the eight neighborhood clusters. From the "bottom", data are pulled from the neighborhoods.
Each neighborhood has a multilevel switch used to route traffic flows between neighborhoods and between the neighborhoods and the central core. These switches and their connections in a 3D environment reveal a double butterfly network configuration, a layer of switches that stand in a hierarchy between the neighborhoods and the central core. There is more bandwidth upstream, that is, towards the central core, and, correlatively, relatively less bandwidth downstream in the neighborhoods.
(5) Double Wishbone 2D Torus Ring Network Structure in 3D NoC
The eight switches in the interior of each neighborhood cluster are connected to a two dimensional torus ring that is on the outside of the central master node. The torus ring is structured in a double wishbone configuration that loops around the central node and connects to each neighborhood switch. There are four switches in the 2D torus ring that connect to the eight neighborhood switches that correspond to the facade of each of the four sides of the cube.
In another embodiment of the present invention, two 2D torus rings are organized around an axis. One ring is ordered at the axis of the plane corresponding to the sides of the 3D SoC cube, while the other ring is structured at the axis of a plane at ninety degrees. The two rings meet at the edges in order to exchange information at their conjoining two switches. This model provides fault tolerance capabilities because if one of the rings is disabled, the system is still operational.
In a further embodiment of the invention, the 2D torus rings are connected to opto-electronic integrated circuits in order to process very high bandwidth communications. The advantage of the opto-electronic circuits integrated into the 3D NoC is maintenance of high bandwidth at the top of the network hierarchy that connects the master node to the neighborhood clusters.
(6) QoS Optimization of 3D NoC Interconnects with Priority Based Model
Quality of service (QoS) techniques are stochastic processes used to ensure high quality solutions to complex networking problems. Specific algorithms, including shortest path and traveling salesman problem (TSP) algorithms, are employed for continuous load balancing. Solving network optimization problems that allocate resources based on changing priorities is a particular challenge of the present system.
Since the present system constantly readjusts its priorities, the speed of data flows is variable. The recalibration of processes according to varied criteria stimulates discontinuous change. The QoS algorithms optimize the modulating network architecture to accommodate plasticity behaviors.
(7) Intranode Multi-Way Router in 3D NoC
In order to maximize efficiency, the present system integrates a router into a layer of each 3D circuit node. Data from each 3D node are sent to the router from interconnects attached to each layer. Data are also received at the intra-nodal router and sent to multiple layers of the 3D IC. In order to accommodate the traffic flows at peak times, the router circuitry has built-in buffers that queue the data packets for orderly traffic flow within the network.
Nodes in different positions of the NoC emphasize the positioning of the routers in different locations within each node. The routers in opposite corners of the 3D NoC cube appear in different locations of their respective node.
Digital routers appear in the center layer of 3D ICs. However, analog components in routers appear on the periphery of 3D ICs because of their noise interference. In these cases, the peripheral layers have shielding to separate these layers from other layers in the 3D node.
(8) Differentiated Bandwidth Interconnects in Hierarchical 3D NoC
The network architecture in the 3D NoC is hierarchical, with higher bandwidth at the top, connecting the neighborhood clusters and the central master node, and relatively lower bandwidth at the bottom, connecting the individual nodes within each neighborhood. The middle layer consists of the eight switch subsystem that connects the top and bottom layers.
The top layer has high bandwidth capabilities, including implementations with opto-electronic switch circuitry. However, the neighborhood clusters generally have far less bandwidth, with interconnects of less width and less capacity among inter-nodal interconnects. The interconnects connecting the nodes to the switch have intermediate capacity, while the interconnects connecting the nodes to each other have smaller capacity. The interconnects and TSVs within a node are far smaller than internodal interconnects.
This network architecture model corresponds to the function of capillaries in natural circulatory systems, or the function of nerves in neural systems, in which the furthest periphery from the core has the narrowest capillaries or neurons.
The logic of the network architecture described here reveals a fail-safe functional advantage because if the pathways of one node are damaged, the system reroutes the network to optimize functionality of the remaining nodes.
(9) Adaptive Routing in Hierarchical 3D SoC Using Shortest Path Optimization for Load Balancing
The present invention uses optimization algorithms to solve MOOPs involving data routing within the neighborhoods and between nodes in the whole network. The system uses hybrid adaptive routing algorithms to accommodate the two main levels of network control. For the intra-neighborhood routing protocols, the network uses minimally adaptive routing techniques, while the system uses fully adaptive techniques for global routing.
Adaptive routing approaches develop strategies that seek to avoid bottlenecks. In order to perform this network flow routing objective, the pathways used by the flow control process are optimized according to shifting priorities.
In the context of the 3D NoC, the system simultaneously optimizes the global routing with the local routing within the neighborhoods in order to maximize load balancing in a continuously recalibrating mechanism.
(10) Scheduled Routing in 3D NoC Using Buffer Modulation, Metadata and Time Multiplexing
A master scheduler in the central core controls data traffic. Other schedulers are positioned in the eight neighborhood switches in order to control intra-neighborhood data traffic flows. The schedulers route priorities in the queues of the switches. The scheduling mechanisms use time-multiplexing to organize the logic of data flows.
A request for a flit is sent by a receiving router to a sending router. The receiving router then schedules traffic flow between the two routers. The two routers exchange credits and debits with the various requests for flit flows. Since the data flow exchange rate is typically variable, the data are buffered in the routers to modulate the flow control process.
Meta-tags are used to mark each data packet. Metadata for each task are read at each location in the flow control path and routed to the appropriate destination. Use of meta-tags is an efficient method of directing traffic to available nodes.
Arbitration scheduling in each switch is used to minimize latency by employing modulating queuing buffers. The scheduled routing of data flows is constantly modified as the demands on the system change and as the available resource supplies vary. In effect, each reservation of scheduling is a temporary reservation that is constantly updated to accommodate the load balancing of the global network.
The use of scheduling flow control by using meta-tags prevents resource conflicts so as to maximize network flow efficiencies.
(11) Opto-electronic Switch for Variable Mesochronous Clock Synchronization in 3D NoC
Mesochronous clock synchronization refers to intermediation between the top and bottom layers in a hierarchical network. In the context of the 3D NoC, the clocks in the neighborhood node clusters use variable timing to modulate their operations to accommodate on-demand functions. The operation of the globally asynchronous locally asynchronous (GALA) model that connects the elements in the network hierarchy occurs in the middle layer. This mesochronous clock synchronization is constantly modulating between the top and bottom layers.
The multilayer mesochronous communication matrix controls the clocking between the top and bottom layers in the 3D NoC by using opto-electronic switches in the 2D torus ring(s). The integrated optical circuit uses an opto-coupler to interact with other optical circuits at the middle layer. The optical network connector transmits and receives data using a light emitting device and a light receiving device. Data are converted into pulses of light in the opto-electronic switch, while a tiny laser-on-chip transmits the light pulses to other opto-electronic switches. The optical signal is adjusted by using on-board amplifiers and attenuators, while a micro-mirror assembly is used to deflect the optical signal.
Though they are high bandwidth devices, the optical switches represent a bottleneck in the network at the input and output sections. The conversion and de-conversion of photonic signals are made by sections of the opto-electronic switch that contain substantial caching.
The combined opto-electronic switches in the 2D torus ring(s) comprise a photonic grid that connects high bandwidth switches with low power interconnects.
In another embodiment of the system, electronic circuitry is used in the switches in the 2D torus ring(s).
Performance Specifications
Bandwidth Specifications in the 3D NoC
EconomyStandardHigh PerformanceAddress Space for128 Gb256 Gb256 Gbeach NodeChannel Width at256 bit wide512 bit wide512 bit wideConnecting NodeschannelschannelschannelsBandwidth 4 Gb/s 8 Gb/s 16 Gb/secThroughput perNodeThroughput at 2D 80 Gb/s160 Gb/s320 Gb/storusTotal System140 Gb/s280 Gb/s560 Gb/sBandwidth
(II) 3D SoC Multi-chip Network Architecture
The 3D SoC networks with other chips to create a scalable high performance computing system. In this sense, the SoC is treated as a node in a computer network. The chip is designed to easily network with other SoCs for macro parallelization.
The present invention organizes networked SoCs to have external access to internal neighborhood clusters and nodes for inter-operational behaviors in a multi-extensive processing environment. In this high performance environment, rather than have eight neighborhoods and a central master node, the system has many neighborhood clusters operating autonomously yet interactively in the larger scalable system.
The present invention describes solutions to problems involving the integration and optimization of cubic SoC's in a parallel scalable computing environment.
(1) Method for Stacking 3D SoCs Using a Cubic Junction Network Connection
One of the advantages of using the cubic configuration of the 3D SoC package is that it may be stacked in larger computing systems.
The 3D SoC employs a cubic junction for networking with other SoCs. The junction connects nine lanes in a parallel switch within an SoC to other SoCs. These nine lanes connect to the eight neighborhood clusters and the central master node. This junction switch splits high bandwidth into nine pipelines in order to directly connect with each neighborhood and the master node. The system uses a fat tree interconnection configuration.
This model allows the direct access of various SoC neighborhoods to other SoC neighborhoods.
The high performance networking system uses optical transceiver circuitry at each neighborhood switch to control external traffic.
In another embodiment of the invention, each SoC neighborhood connects wirelessly to other SoCs using high-bandwidth RF technology.
(2) System and Method for Linking Multiple 3D SoC Nodes in Point-to-point Internodal Network
The system is further organized in a network configuration that allows internodal communications. With hundreds of SoCs, thousands of nodes are organized as autonomous units. By numbering the nodes and organizing reconfigurable clusters of 3D circuits, the system accesses various nodes in a point-to-point configuration.
The nine pipeline junction connecting the chips allows access to each chip's double-wishbone 2D torus ring(s), which feed directly to specific neighborhood clusters. These pipelines then access individual nodes in each neighborhood. This point to point network connection configuration allows a more accessible strategy than nearest neighbor connection configurations.
The advantage of this connection configuration model is that it allows the direct interoperation of nodes beyond the neighborhood level. This is important particularly because the neighborhood configurations periodically change.
(3) System and Method for Autonomous Organization of 3D SoC in Scalable Parallel Computer Networks
The 3D SoC is an intelligent chip because it contains reconfigurable, reprogrammable and auto-programmable features. When combined in highly parallel network systems, the SoCs programmability features produce a highly adaptive computer system capable of multi-petaflop performance.
Performance Specifications: High Performance Computing
EconomyStandardHigh PerformanceNumber of8 × 8 × 8 = 51212 × 12 × 12 = 172824 × 24 × 24 = 13,824iSoCs in cubeNumber of cubes888Total number409613,824110,592of iSoCs inSystemTotal number143,360967,6807,741,440of Nodes1.35 TFlop/s per 5.53 PFlop/s 18.66 PFlop/s149.3PFlop/sChip2 TFlop/s per Chip8.192 PFlop/s27.648 PFlop/s221PFlop/s10 TFlop/s per40.96 PFlop/s138.24 PFlop/s1ExaFlop/sChip
Although the invention has been shown and described with respect to a certain embodiment or embodiments, it is obvious that equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In particular regard to the various functions performed by the above described elements (components, assemblies, devices, compositions, etc.) the terms (including a reference to a "means") used to describe such elements are intended to correspond, unless otherwise indicated, to any element that performs the specified function of the described element (i.e., that is functionally equivalent), even though not structurally equivalent to the disclosed structure that performs the function in the herein illustrated exemplary embodiment or embodiments of the invention. In addition, while a particular feature of the invention may have been described above with respect to only one or more of several illustrated embodiments, such feature may be combined with one or more other features of the other embodiments, as may be desired and advantageous for any given or particular application.
Acronyms
    3D, three dimensional    ASIC, application specific integrated circuit    BOOP, bi-objective optimization problem    CMOS, complementary metal oxide semiconductor    CPLD, complex programmable logic device    D-EDA, dynamic electronic design automation    DIVA, data intensive architecture    DLP, data level parallelism    EDA, electronic design automation    EHW, evolvable hardware    eMOOP, evolvable multi-objective optimization problem    Flops, floating operations per second    FPCA, field programmable compute array    FPGA, field programmable gate array    GALA, globally asynchronous locally asynchronous    HPPS, high performance processing system    ILP, instruction level parallelism    IP, intellectual property    iSoC, intelligent system on a chip    MEMS, micro electro mechanical system    MONARCH, morphable networked micro-architecture    MOOP, multi-objective optimization problem    MPSOC, multi-processor system on a chip    NEMS, nano electro mechanical system    NoC, network on a chip    PCA, polymorphous computing architecture    PIM, processor in memory    QoS, quality of service    RISC, reduced instruction set computing    SCOC, supercomputer on a chip    SoC, system on a chip    SOI, silicon on insulation    SOPC, system on a programmable chip    SPE, synergistic processor element    TLP, thread level parallelism    TRIPS, Tera-op reliable intelligently adaptive processing system    TSV, through silicon via    ULSI, ultra large scale integration    VLSI, very large scale integration    WSPS, wafer level processed stack packages

This patent is subject to a terminal disclaimer.

Interconnect architecture in three dimensional network on a chip