WO2014070149A2 - Procédés d'identification de substance à l'aide de remembrement - Google Patents

Procédés d'identification de substance à l'aide de remembrement Download PDF

Info

Publication number
WO2014070149A2
WO2014070149A2 PCT/US2012/062629 US2012062629W WO2014070149A2 WO 2014070149 A2 WO2014070149 A2 WO 2014070149A2 US 2012062629 W US2012062629 W US 2012062629W WO 2014070149 A2 WO2014070149 A2 WO 2014070149A2
Authority
WO
WIPO (PCT)
Prior art keywords
pools
wells
screening
column
pool
Prior art date
Application number
PCT/US2012/062629
Other languages
English (en)
Other versions
WO2014070149A3 (fr
Inventor
Keith E. Stormo
Quanzhou Tao
Robert H. BOGDEN
Evan K. HART
Original Assignee
Amplicon Express, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amplicon Express, Inc. filed Critical Amplicon Express, Inc.
Priority to PCT/US2012/062629 priority Critical patent/WO2014070149A2/fr
Publication of WO2014070149A2 publication Critical patent/WO2014070149A2/fr
Publication of WO2014070149A3 publication Critical patent/WO2014070149A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • This application pertains to substance identification methods using pooling.
  • Pooled biological material such as DNA, RNA, proteins, and the like, may be screened by a wide variety of methods, such as sequencing, PCR (Polymerase Chain Reaction), DNA/DNA hybridization, DNA RNA hybridization, RNA/RNA hybridization, single strand DNA probing, protein/protein hybridization, and a wide variety of additional methods.
  • references describing many of these methods include Ausubel et.al., "Short Protocols in Molecular Biology,” Wiley and Sons, New York and Sambrook et.al, "Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Press, New York, as well as numerous others. Also referenced are U.S. Pat.
  • FIG. 1 is an entire BAC Library comprised of BAC clones in individual wells of 120, 384-well plates, designated as Superpools 1 -10 (SP1 -10).
  • FIG. 2 is the Library Code Superpool Collection plate copy #1 having 96 wells.
  • FIG. 3A is the SP1 with 12 plates having a clone address in plate #8.
  • FIG. 3B is the SP1 with 16 rows having a clone address in row #4.
  • FIG. 3C is the SP1 with 24 columns having a clone address in column #16.
  • FIG. 3D is the SP1 with 24 diagonals having a clone address in diagonal #6 as specified in table 4.
  • FIG. 4 is the small high resolution pools of the plate, row, column, and diagonal samples of DNA, cDNA or proteins with a positive and negative control in wells E2 and F2 respectively.
  • FIG. 5 is the further re-pooling of a subset of the wells of FIG. 4 onto the Matrix Pool Plate or specific repooling designs in a Plate, Row, Column, and Diagonal matrix loading pattern.
  • FIG. 6 is a grid representing 384 wells of a plate.
  • FIGS. 7 and 8 are grids representing 384 wells of logical arrays.
  • FIGS. 9a and 9b combined is a grid representing wells of a logical array including five 384-well plates; FIG 9b is a continuation of 9a on another page.
  • FIG. 10 is a grid representing one of the plates in FIGS. 9a and 9b.
  • FIGS. 1 1 and 12 are grids representing wells of a logical array including twelve 384-well plates.
  • FIGS. 13 and 14 are grids representing 384 wells of a logical array.
  • the embodiments herein allow the incorporation of "loss-less information compression and error correction” or other known error correction strategies to increase the robustness of identification with significantly reduced numbers of samples to be processed by the end user. By having the samples pooled again after collection, it is possible to drastically reduce manipulations by the end user while still keeping very fine detail in the identification of the individual samples or populations originally pooled. Error-correction methods are well known in the computer data transmission field, but have not been used in the pooling of biological or chemical samples. Such methods allow a large reduction in the number of experiments to identify the specific biological sample or population containing a region of interest. [0019] The embodiments herein include screening methods where the entire "pooling strategy" used is determined a priori. It is possible to conceive strategies where the strategy used in subsequent levels of processing depends on the outcome of previous levels, and such methods may increase efficiency.
  • the pooled material can be from individuals or a population.
  • the pooling of small, high resolution pools in a matrix allows for a lower number of samples to be analyzed.
  • the resulting high resolution data obtained from screening matrix pools are equivalent to the data obtained if the researcher had analyzed the complete set of small pools (much more expensive, time consuming, and difficult).
  • the embodiments also give the added advantage of having two positive signals for identification. This reduces errors associated with a false positive when only one signal is obtained for identification, as in known methods.
  • the matrix pooling can be just for one superpool. Alternatively, it can be a matrix of a variety of different superpools and/or across a variety of different types of pools to allow the screening of the complete library with just one round of experiments. To do this, each small pool would be added to between 6 and 20 of the collection of re-pooled intermediate or final pools. Then, with the total number of pools of between 40 and 100, the complete library (or any set of biological samples) could be screened with high confidence and the ability to resolve multiple hits. If the library had a large redundancy of signal, the total number of pools could be increased to maintain accurate resolving power of the matrix method.
  • the incorporation of positive controls in a matrix pattern can be used for quality assurance and for assisting in deconvolution if desired.
  • Biological materials may include Bacterial Artificial Chromosome (BAC) genomic DNA libraries and other biological or chemical libraries like cDNA libraries, protein libraries, RNA libraries, DNA libraries, cellular metabolic libraries, and chemical libraries.
  • BAC Bacterial Artificial Chromosome
  • the current state of the art in pooling of biological materials for screening includes collecting all of the indexed microtiter plates containing the BAC library and then stacking these plates into a cube. These indexed plates are generally 96, 384, 864 well, or sometimes even 1536 well microtiter plates.
  • the cube is then transected by a number of different planes (usually 4 to 8) which produce a large number of pools from each plane.
  • the collection of all the pools from all of the planes is then screened to identify the clones of interest.
  • This scheme is the current state-of-the-art and can identify multiple clone hits with some degree of reliability to identify multiple targets (i.e., BAC clones) at a specific coordinate.
  • Embodiments herein may provide beneficial accuracy, efficiency and reduced cost by using at least one additional step of repooling the intermediate subpooled genomic DNA clone DNA into a final screening pool.
  • the individual genomic DNA clone may be in at least three unique final screening pools, such as from three to ten unique final screening pools, or from four to eight.
  • BAC Library is from an organism with a genome larger than 1 ,000 megabases (Mb, millions of base pairs)
  • the researcher may find that there are very few ambiguous hits in a plate, row, column, and diagonal (PRCD) plate.
  • the Plate, Row, and Column pools correctly identify the clone of interest without the need for the Diagonal Pools. If the Diagonal Pools are only screened to solve the infrequent ambiguity, there would be a reduction in the number of PCR experiments.
  • a Bac-Bank is a way of storing fragments of DNA, together constituting the whole genome of an organism.
  • the DNA of an organism is (semi) randomly cut in pieces, and these fragments are inserted into bacteria, which are then plated out so that a single colony grows from a single modified bacterium. Only modified bacteria are allowed to grow by using a bacterium that is potentially resistant to a certain antibiotic, and whose resistance is "switched on” by the presence of a foreign DNA fragment (insert), and by using a growth medium containing the antibiotic. The resulting (potentially) unique colonies of bacteria are then picked up individually and transferred to the wells of 384-well plates.
  • the resulting stack of plates holding a large number of unique bacteria, ideally containing the whole genome of the original organism, is known as a "Bac-Bank". It serves as a research database of the genome of the original organism. This database can be searched for fragments of DNA using PCR techniques.
  • Pooling is a method that allows one to quickly and economically search a Bac-Bank for the presence of certain DNA fragments.
  • a Bac-Bank normally contains a large number of clones (-100,000). Testing all clones individually for the presence of a fragment of DNA occurring only a few times (typically less than 100 times) in the original organism's genome is prohibitively expensive and laborious.
  • pooling the DNA of several clones is gathered into a much lower number of wells (pools), every well containing DNA from several clones and every clone's DNA being present in multiple wells.
  • the distribution pattern (“pooling method,” “pooling strategy,” or”rule-set”) is designed in such way that, when using PCR reactions to screen the pools, a pattern (of PCR reaction results) emerges that may be unique to the clone(s) having the required properties.
  • a simple example take a 384-well plate having 16 rows of 24 columns; imagine pooling all wells horizontally and vertically, resulting in 16 row-pools and 24 column pools. If a single clone in this plate has a certain property, only the column-pool and the row-pool that particular clone is in will display a positive reaction when screened; the other 38 pools will be negative. Using only 40 PCR reactions it is therefore possible to pinpoint the positive clone in this 384-well plate; almost a tenfold reduction in labor and cost. As long as there are relatively few individuals with a certain property there are few errors. For properties that are shared among many individuals pooling methods may break down (yield incorrect results, either false-positives or (worse) false-negatives), and when this happens one has to resort to screening the clones individually.
  • Bac-Bank Most often the individual clones in Bac-Bank are identified/labeled according to some hierarchical structure dictated by the physical properties of the Bac-Bank. The number of dimensions of a Bac-Bank is then related to the hierarchical structure of the storage format.
  • Bac-Bank An example: the clones of a Bac-Bank are individually stored in wells on a plate. The wells are arranged in a rectangular pattern of rows and columns. If the plate constitutes the whole Bac-Bank, then the Bac- Bank can be viewed as one-dimensional if all the wells on the plate have consecutive numbers from left to right and top to bottom. One single parameter (well number) suffices to address every individual clone/well on the plate, and therefore the Bac-Bank is one-dimensional. A more natural approach in this example would be to address each well by its column and row numbers; then we would need two parameters to address an individual well, and therefore the same Bac-Bank can be two-dimensional as well.
  • Embodiments herein are distinguished from known methods in that a collection of substances is systematically divided into smaller subsets which are then re-pooled to make the final screening pools.
  • the pooled material can be from individual samples or a population of samples.
  • the pooling of high resolution small pools in a matrix allows for a lower number of user experiments to have higher resolution (as if the researcher had analyzed the complete set of small pools).
  • One embodiment includes a two-step method that first screens for a superpool in which an item of interest appears. Then, that specific superpool's pools are re-pooled into matrix pools (which are 36 matrix pools instead of 76 pools).
  • matrix pools screened in this method also give the added advantage of having two or more positive signals for identification. This reduces the current state-of-the-art limitations associated with a false positive and/or false negative experimental result when only one signal is obtained for identification.
  • the Round I PCR may be performed on all of the Superpools containing all BAC clones in the Library. Each Superpool may contain 4,608 individual BAC clones.
  • the results from Round I of PCR identify Superpool BAC clone(s) with the sequence of interest (there may be more than one Superpool identified). The researcher may choose to pursue one or more positive hits from the Round I PCR.
  • the Round II PCR may be performed on the Matrix Pools for the specific Superpool identified in Round I PCR.
  • Round II PCR uses 36 PCR experiments plus controls (for each positive hit pursued from Round I PCR).
  • the results from Round II PCR allow the researcher to identify the plate and well position for several positive hits and to rule out many potential false positives (in the particular Superpool(s) being pursued).
  • Round II PCR screening of PRCD pools requires 76 PCR reactions plus controls.
  • the Matrix system reduces the PCR experiments by 50%.
  • the Matrix Pools are PRCD pools combined so that EACH of these PRCD pools is contained in TWO unique Matrix Pools. There are a total of 36 Matrix Pools for each Superpool. Eight Matrix Plate Pools (MPP), eight Matrix Row Pools (MRP), 10 Matrix Column Pools (MCP) and 10 Matrix Diagonal Pools (MDP). There are at most 1 ,152 individual BAC clones inside each Matrix Pool well.
  • MPP Matrix Plate Pools
  • MRP Matrix Row Pools
  • MCP Matrix Column Pools
  • MDP Matrix Diagonal Pools
  • the matrix pooling can be just in one superpool. Alternately, it can be a matrix of a variety of different superpools and/or across a variety of different types of pools to allow the screening of the complete library with just one round of experiments. To do this, each small pool may be combined with any number (generally between six and many thousands depending on the sensitivity/robustness of the user's experimental screening strategy) of final collection pools (which are re-pooled intermediate pools). For this example we'll use the range of between 6 and 20 collection pools (fully compatible with a PCR based screening technology).
  • FIGS. 1 -5 are a graphical representation of an embodiment.
  • Example 2 Tables 6 and 7 are alternate embodiments of FIG. 3D and FIG 4 respectively.
  • Tables 8-11 represent additional alternative embodiments of specific repooling designs as depicted in FIG. 5.
  • FIG. 1 represents ten Superpools of the entire BAC Library 10 containing 120, 384 well-plates 2 stacked on top of each other in ten sets of twelve plates. A wide, almost limitless set of indexed microtiter plates may be used for plates 2.
  • FIG. 1 is a combined stack of ten Superpools (SP1 -SP10). Each Superpool has a stack of twelve plates 2 stacked upon each other. The plates could be any multi-well unit that can be arranged into a hierarchical structure. Claimed herein are 96, 384, 864, and 1536-well units.
  • a 96-well Superpool Plate 20 comes with a positive (at A1 ) control 4 and a negative (at B1 ) control 6 and a sample from individual Superpools 8.
  • Superpool plate 20 provides the template for at least 800 PCR experiments. After receiving Round I PCR gel electrophoresis results, the researcher determines which Superpool to screen for Round II PCR.
  • any of Superpools SP-1 to SP-10 may be separated into pools of plates, rows, columns, and diagonals, which are all based on the hierarchical structure for the clone of interest to allow the researcher to find the specific coordinate or unique address of the well position with the clone of interest.
  • At least three of these four hierarchical structures may be used or any combination of three of the four hierarchical structures to insure or guarantee finding the specific coordinate well position with the clone of interest through iteration or redundancy (e.g., FIG. 3D diagonal pool, plus FIG. 3A plate pool, plus FIG. 3C column pool).
  • FIGS. 3A-3D represent a Superpool with a stack of 12 plates with 384 wells.
  • FIGS. 3A-D use four different search patterns to find the precise well having the clone of interest.
  • FIG. 3A identifies the plate of interest in plate pool 30 of SP1 , e.g., plate P-8.
  • Fig. 3B identifies the row of interest in row pool 40 of SP1 , e.g. row R-4.
  • FIG. 3C identifies the column of interest in column pool 50 of SP1 , e.g. column C-16.
  • FIG. 3D identifies the diagonal of interest in diagonal pool 60 of SP1 , e.g. diagonal D-6.
  • FIG. 4 depicts the intermediate subpools in the hierarchical structure plate pool 30, row pool 40, column pool 50, and diagonal pool 60 that were generated by processing individual subpools to extract the material according to the hierarchical structure.
  • FIG. 4 is where the isolated material from the subpools is stored in a stable form before repooling the intermediate subpooled material into the Final Screening Pools, as shown in FIG. 5.
  • FIG. 5 represents the repooling of the intermediate subpooled material into a number of final screening pools based on a specific repooling design, wherein individual information is in at least three final screening pools, or at least four final screening pools and no more than eight final screening pools.
  • plate 80 of final screening pool materials is screened, the specific coordinates are determined which allows the identification of the well position of the clone of interest.
  • This description is based on 384 well index plates, but it could be used with other plate formats as well with appropriate considerations. It is also based on a BAC genomic DNA library comprised of individual BAC clones, but it could be used with a large variety of biological sample collections or chemical sample collections.
  • the system includes a collection of multiple Superpools that are screened during First Round PCR, to determine which set of Matrix Pools to screen during Second Round PCR. The total number of Superpools is determined by the total number of clones in the BAC library. Each Superpool has its own 96-well plate of corresponding Matrix Pools.
  • Each superpool includes twelve consecutive 384-well plates from a BAC library. DNA is prepared by growing EACH BAC CLONE separately (to avoid growth competition between BAC clones) then combining the 4,608 cultures into one large-scale BAC prep. The Superpool of BAC DNA is then aliquoted onto a 96- well plate. Superpool SP-1 has all the BAC clones in the first twelve plates of the BAC library (Plate 001 to Plate 012). Superpool SP-2 has all the BAC clones in the second twelve plates of the BAC library (Plate 013 to Plate 024). This naming continues for the entire library.
  • Matrix Pools For each superpool there is one set of Matrix Pools (this set of 36 Matrix Pools are aliquoted onto a Matrix Pool Plate.
  • the Matrix Pools of Superpool SP1 are named Matrix Plate Pools, Matrix Row Pools, Matrix Column Pools, and Matrix Diagonal Pools.
  • Matrix Plate Pools 1 MPP-A1 through 1 MPP-H1 for the 8 wells that contain the matrix of plates 1-12 in Superpool SP1.
  • Each Matrix Plate Pool contains 1 ,152 clones.
  • Table 1 indicates the clones in each well.
  • FIG. 5 shows plate repool 31 , which also describes the content of the Matrix Plate Pools, namely, the plate numbers of the clones in respective Matrix Pools. The same process is repeated for as many superpools as are needed for the complete library.
  • Matrix Row Pools 1 MRP-A2 through 1 MRP-H2 for the 8 wells that contain the matrix of rows A-P in Superpool SP1.
  • Each Matrix Row Pool contains 1 ,152 clones for twelve 384 well plates.
  • Table 2 shows the composition of each well in the Matrix Row Pools.
  • FIG. 5 shows row repool 41 , which also describes the content of the Matrix Row Pools, namely, the row letters of the clones in respective Matrix Pools.
  • Table 3 shows the composition of each well in the Matrix Column Pools.
  • FIG. 5 shows column repool 51 , which also describes the content of the Matrix Column Pools, namely, the column numbers of the clones in respective Matrix Pools. TABLE 3.
  • the diagonal pools are a collection of clones from all twelve plates in one superpool that has been transected by a plane that goes diagonal in an XY plane and diagonal in a XZ plane through the 12 plates. The diagonals are named by the number of the column that the clone from row A on plate 1 of the specific diagonal.
  • Table 4 shows the exact location by plate number, row letter, and column number of each well included in each diagonal pool. Notably, as the diagonal number (column number) approaches 24, the diagonal pool wraps back to column 1 for a 16 row by 24 column plate.
  • Diagonal pool composition is depicted graphically by FIG. 3D and corresponds to the construction of diagonal pool 60 in FIG.4.
  • Table 5 shows the composition of the Matrix Diagonal Pools.
  • FIG.5 shows diagonal repool 61 , which also describes the content of the Matrix Diagonal Pools, namely, the diagonal numbers of the clones in respective Matrix Pools.
  • Table 4 is but an example of a diagonal scheme that is non-redundant with other pools.
  • the embodiments are not limited to one specific diagonal scheme since there are additional diagonal scheme that can be used as alternatives to this diagonal scheme.
  • the identity of a specific positive clone from the library can be determined.
  • the specific identification can be determined by a number of ways. If the pool design and matrix design are written or available in electronic form, the unique clone can be identified by a visual or electronic search. There can also be algorithms written based on the pool and matrix designs that can identify the unique clone.
  • the second example describes a method to form a matrix of a variety of different superpools and/or across a variety of different types of pools to allow the screening of the complete library with just one round of experiments.
  • each small pool or subpool would be added to between 6 and 20 of the collection of re- pooled intermediate or final pools.
  • the complete library could be screened with high confidence and the ability to resolve multiple hits. If the library had a large redundancy of signal, the total number of pools could be increased to maintain accurate resolving power of the matrix solution.
  • Note: 94 experiments is a convenient number, because current screening technologies are performed on a 96-well index plate format (94 experiments will allow room for a positive control and negative control).
  • Example 2 further illustrates the benefits and possibilities of the embodiments. This example is also based on 384 well index plates, but it could be used with other plate formats as well with appropriate considerations. It is also based on a BAC genomic DNA library comprised of individual BAC clones, but it could be used with a large variety of biological collections. The superpools will be composed of eight 384 well plates per superpool and with 10 superpools combined into one large set of matrix pools.
  • the individual superpools are numbered so that each individual 1/3 plate, row, column and diagonal pool has a unique number. Since there are 88 pools per superpool and ten superpools in this example, there are a total of 880 individual pools that will be combined into one large set of matrix pools.
  • the idealized degree of redundancy can dramatically improve the ability to identify multiple positive clones in one screening and thus minimize ambiguous results (when the user is analyzing data from the screening experiments).
  • the first 1/3 plate pools are formed by collecting all of the clones in plate 1 from columns 1-8. Then the second 1/3 plate pool is all of the clones from columns 9-16 of plate one. This continues on until the 24th 1/3 plate pool is from columns 17-24 of plate 8. The twenty-four 1/3 plate pools from superpool two would be considered being in pools 89-112 and so on until the tenth superpool where the 1/3 plate pools would be in pools 793-816.
  • the row pools would be built the same way as Example 1 but since there are only 8 plates in each superpool, each pool would have 192 clones. All of the clones in row A of the eight plates would be pooled together and these clones would be considered pool number 25. This would continue on in a similar fashion so all of the clones in row B of all eight plates of the superpool would belong to pool 26 (and so on) until finally, the pool of all of the clones in row P of the first eight plates would belong to pool number 40. Similarly, the row pools from the second superpool will be in pools numbered 113-128. This would continue in a similar fashion until all of the superpool individual clones belong to row pools and each are assigned unique numbers.
  • the column pools would be formed the same way as in Example 1 but since there are only 8 plates in each superpool, each pool would have 128 clones. All of the clones in column 1 of the eight plates would be pooled together and would belong to pool number 41. This would continue on in a similar fashion until all of the clones in column 2 of all eight plates of the superpool would belong to pool 42 (and so on). Until finally, the pool of all of the clones in column 24 of the first eight plates belong to pool number 64. Similarly, the column pools from the second superpool will be in pools numbered 129-152. This would continue in a similar fashion until all of the superpools belong to column pools and each are assigned unique numbers.
  • the diagonal pools would be formed the same way as in Example 1 but since there are only 8 plates in each superpool, each pool would have 128 clones. See Table 6 for the 8 plate superpool diagonal composition. All of the clones in diagonal 1 of the eight plates would be pooled together and would belong to pool number 65. This would continue on in a similar fashion until all of the clones in diagonal 2 of all eight plates of the superpool would belong to pool 66 (and so on). Until finally, the pool of all of the clones in diagonal 24 of the first eight plates belong to pool number 88. Similarly, the diagonal pools from the second superpool will be in pools numbered 152-176. This would continue in a similar fashion until all of the superpools belong to diagonal pools and each are assigned unique numbers.
  • Table 7 is designed for 88 pools in each subset (superpool) and ten subset (superpools) in the complete set. These unique pool numbers are used to construct various tested screening pool pooling strategies. Notably, as the column number approaches 24, the diagonal pool wraps back to column 1 for a 16 row by 24 column plate.
  • Table 6 describes an alternate embodiment for constructing the diagonal pool composition for an 8 plate Superpool.
  • Table 7 sequentially assigns numbers to individual small pools or subpools from ten consecutive from eight plates so that the subpools may be repooled into final screening pools according to example alternative embodiments depicted in Tables 8-1 1.
  • Tables 8-1 1 describe various embodiments in the systematic or randomization of the loading of the small pool or subpooled plate, row, column, and diagonal pooled DNA (FIG. 4) into an alternate Matrix Pool Plate format (FIG. 5).
  • Tables 8, 9, 10 and 11 show four of the many specific repooling designs that were tested to demonstrate the utility of this patent.
  • Tables 12-16 are data showing multiple embodiments of various randomization schemes for pooling a quantification of data loaded into the Matrix Pool Plate (FIG. 5).
  • Table 12 Summary of various screening pool design unique clone identification. Pooling Summary with each clone contained in 4 to 8 unique pools.
  • Table 13 Summary of various screening pool design unique clone identification.
  • Table 14 Summary of various screening pool designs searching for one unique clone identification.
  • Table 15 Summary of various screening pool designs searching for two unique clone identifications.
  • Tables 13, 14, 15 and 16 show data collected from various pooling designs.
  • pooled biological material such as DNA, RNA, proteins and the like
  • methods such as sequencing, PCR (Polymerase Chain Reaction), DNA/DNA hybridization, DNA/RNA hybridization, RNA/RNA hybridization, single strand DNA probing, protein/protein hybridization, and a wide variety of additional methods.
  • Construction of pools and superpools for screening as described herein differs from known methods in that the biological material set is systematically divided into a variety of smaller subsets, which are then re-pooled to make the final screening pools.
  • This pooled material can be from individual samples or a population of samples. In order to reduce the analysis time, materials, and expense, the pooling of high resolution small pools in a matrix allows for a lower number of user experiments to have higher resolution (as if the researcher had analyzed the complete set of small pools).
  • a substance identification method includes using a collection of segregated substances placed in respective wells of a plurality of collection plates physically or logically arranged in a stack.
  • the wells are arranged in a plurality of rows and a plurality of columns and individual substances have a unique coordinate locating a well position defined by a plate identifier, a row identifier, and a column identifier.
  • the method includes combining the substances into four or more intermediate subpools in respective wells of a subpool plate.
  • the four or more intermediate subpools are of at least one type of intermediate subpool.
  • One to four of the types of subpool are selected from the group consisting of a plate pool from wells having a common plate identifier, a row pool from wells having a common row identifier, a column pool from wells having a common column identifier, and a diagonal pool from wells having column and/or row identifiers per plate that are offset with respect to column and/or row identifiers per plate of any adjacent plate in the stack.
  • the four or more intermediate subpools are repooled into a number of final screening pools less than the four or more intermediate subpools.
  • the final screening pools are placed in respective wells of a matrix pool plate based on a repooling design providing the subpooled substances in at least three different final screening pools.
  • the method includes screening the final screening pools and identifying the presence of an item of interest associated with a substance. By using the repooling design, the coordinate is determined locating the well position in the collection for the substance associated with the item of interest.
  • the subpooled substances may be different.
  • the collection may be a portion of a BAC library, or may be an entire BAC library.
  • Other possibilities for the substances may be selected from the group consisting of biological material clones or fragments, expressed proteins, purified proteins, materials exhibiting biological activity, chemicals expressed in biological processes, and combinations thereof.
  • the item of interest may be selected from the group consisting of a nucleotide sequence in a biological material clone or fragment, a biological activity exhibited by a material, a chemical composition, and combinations thereof.
  • Biological activity for proteins could include binding to specific chemicals, receptor sites, or antibodies, regulating proteins for transcription or translation, DNA binding proteins that turn other genes on or off, etc.
  • the substances may be biological material clones including genomic DNA clones and the item of interest may be a DNA nucleotide sequence in a genomic clone DNA insert.
  • the method may further include culturing the collection of clones, producing respective individual clone cultures, and forming the intermediate subpools using the individual clone cultures.
  • Biological material fragments may be isolated from the four or more intermediate subpools and stored in a stable form prior to the repooling.
  • the at least one type of intermediate subpool may include four types of subpool including the plate pool, the row pool, the column pool, and the diagonal pool.
  • the offset column and/or row identifiers of the diagonal pool may be offset by one column and/or row with respect to adjacent plates and might not be repeated in the diagonal pool for any other plate.
  • the screening may be selected from the group consisting of sequencing, PCR probing, DNA to DNA hybridization probing, RNA to DNA probing, protein to protein probing, antibody to protein probing, DNA to protein probing, RNA to protein probing, chemical compound to protein probing, ligand to protein probing, and combinations or modifications thereof.
  • the repooling design may provide the subpooled substances in four to eight of the final screening pools to establish the benefits enumerated above.
  • the combining of substances may use four types of intermediate subpools to provide four-dimensions of intermediate subpools.
  • a sum of the plurality of plates, the plurality of rows, and the plurality of columns may be less than a number of the intermediate subpools sufficient to identify the well position of any substance in the array.
  • the repooling design may produce a number of final screening pools sufficient to identify the well position of any substance in the array, even though the number of final screening pools is less than the sum.
  • a method for identifying an individual genomic clone DNA insert from a collection of genomic DNA clones includes the following features.
  • the individual genomic DNA clones are arrayed in a plurality of respective wells of a plurality of collection plates comprised of rows and columns.
  • Individual genomic DNA clones have a specific coordinate locating a well position defined by three or four pools chosen from the group consisting of a plate pool, a row pool, a column pool, and a diagonal pool.
  • the pools are in a hierarchical structure that is composed of a plate identifier, a row identifier, and a column identifier.
  • the method includes culturing the collection of genomic DNA clones and constructing at least four intermediate subpools by combining individual genomic DNA clone cultures in accordance with the hierarchical structure.
  • Genomic DNA clone DNA is isolated from the at least four intermediate subpools and stored in a stable form.
  • the at least four intermediate subpools are repooled into a number of Final Screening Pools based on a chosen repooling design.
  • the subpooled individual genomic DNA clone DNA is in at least 4 Final Screening Pools and no more than 8 Final Screening Pools.
  • the number of Final Screening Pools is screened for a DNA sequence of interest, determining the specific coordinate using the chosen repooling design and identifying the well position of the DNA sequence of interest.
  • a substance identification method includes using a collection of segregated substances placed in respective wells physically or logically arranged in a two-dimensional array.
  • the wells are arranged in a plurality of rows and a number of columns that is at least 1.5 times the plurality of rows.
  • Individual substances have a unique coordinate locating a well position defined by a row identifier and a column identifier.
  • the method includes combining the substances into a number of screening pools in respective wells of a matrix pool plate.
  • a plurality of individual screening pools include substances from wells having a row identifier in common with one other well. Pools are based on a pooling design that provides the pooled substances in two different screening pools.
  • the method also includes screening the screening pools and identifying the presence of an item of interest associated with a substance.
  • the pooling design is used to determine the coordinate locating the well position in the collection for the substance associated with the item of interest.
  • the number of columns may be at least two times the plurality of rows, or at least two times the plurality of rows plus one.
  • the number of screening pools may match the number of columns. Given that the array may be logically arranged, instead of physically arranged, the array may reside on a plurality of microtiter well plates and at least some of the screening pools may extend across a plurality of the plates.
  • the pooling design may provide screening pools from contiguous wells or, instead, one or more of the wells in a pool may be non-contiguous.
  • contiguous wells are those that are adjacent in the sense that they are not separated from one another by another well, whether in a horizontal, vertical, or diagonal direction.
  • a number of the screening pools sufficient to identify the well position of any substance in the array may be less than a sum of the plurality of rows and the number of columns.
  • the pooling design may reduce the two-dimensional array to a one-dimensional array.
  • the one- dimensional array may include pseudo-column pools from a plurality of wells having a common column identifier and another equal number of wells having a row identifier in common with the plurality of wells.
  • the one- dimensional array may include bi-diagonal pools from a plurality of wells that do not have row or column identifiers in common and another equal number of wells having a row identifier in common with the plurality of wells.
  • the other number of wells in the pseudo-column pools or the bi-diagonal pools might not have row or column identifiers in common.
  • the pooling design may instead reduce a part of the two-dimensional array to a one-dimensional array.
  • the screening pools from another part of the two-dimensional array may form screening pools in a second dimension, such as a row dimension.
  • the number of screening pools may be at least the number of wells of the matrix pool plate, as shown Table 17 below, that corresponds to a total number of wells in the pooling design.
  • the combination function formula below Table 17 used to calculate the contents of Table 17 may be used to determine any number of screening pools not shown in Table 17.
  • the method may include culturing the collection of clones.
  • the method may further include producing respective individual clone cultures, forming the screening pools using the individual clone cultures, and isolating biological material fragments from the screening pools.
  • the isolated fragments may be stored in a stable form prior to the screening.
  • FIGS. 6 and 7 demonstrate one example of a substance identification method implementing some of the features of the immediately preceding embodiment.
  • FIG. 6 shows a grid representing wells of a 384-well plate with 24 columns and 16 rows. Accordingly, the number of columns is 1.5 times the number of rows.
  • the physical arrangement of a 384-well plate could be used, benefits exist to using a logical arrangement for the array placed in the same 384-well plate as in FIG. 6, but with the number of columns at least two times the number of rows, or two times the number plus one.
  • FIG. 7 shows a grid representing 384 wells, which may be located in the 384-well plate of FIG. 6. Note that individual well numbers are shown in FIG. 6 sequentially numbered from left to right and from top to bottom. Well numbers in FIG. 6 represent the same wells with a corresponding number shown in FIG. 7. The wells in FIG. 7 are sequentially numbered the same as in FIG. 6 from left to right out to column 24 and from top to bottom down to row M. However, wells 325 to 336 appearing in row N between columns 13 and 24 of FIG. 6 become column 25 for the logical arrangement of FIG. 7. Wells 337 to 384 are similarly shifted in the logical arrangement to form columns 26- 29 of FIG. 7.
  • the substances from the wells may be combined into 29 screening pools, matching the number of columns in FIG. 7.
  • Individual screening pools include substances from wells having a row identifier in common with one other well based on a pooling design that provides the pooled substances in two different screening pools.
  • FIG. 7 shows 28 lightly shaded wells, each of which have a row identifier in common with one other lightly shaded well.
  • Such wells include wells in column 1 and a diagonal extending upward from well 314 in row N, column 2 to well 15 in row A, column 15.
  • any of the wells in columns 2-29 of FIG. 7 could be selected instead of the lightly shaded diagonal of wells to provide wells with a row identifier in common with the row identifier of wells in column 1.
  • the selection could even be random, as long as a given well was not selected for more than two screening pools. However, selecting contiguous wells as often as possible for the screening pool facilitates efficiency in robotic movements of devices collecting substances from the wells.
  • a pseudo-column pool may be created that includes column 1 and "extends" the column through the two-dimensional array in FIG. 7 to include additional wells that have a row identifier in common with the wells of column 1.
  • the FIG. 7 pooling design could be described as including wells having a common column identifier (column 1 ).
  • the other equal number of wells in FIG. 7 has a row identifier in common with the plurality of wells but does not have a row or column identifier in common among the other equal number of wells. That is, none of the wells in the diagonal have a row or column identifier in common with one another.
  • a second screening pool may have the same form as the lightly shaded wells in FIG. 7 but shift to the right one position.
  • screening pool 2 includes the wells of column 2 and a diagonal extending upward from well 315 at row N, column 2 to well 16 at row A, column 16.
  • both screening pool 1 and screening pool 2 include well 314.
  • well 314 is the only "intersection" of those two screening pools. The selection of screening pools may continue, shifting over one position for each pool until all 384 wells are included in screening pools.
  • FIG. 7 also shows 26 darkly shaded wells that may be included in screening pool 15 in like manner.
  • FIG. 7 shows an intersection of the lightly shaded screening pool 1 and the darkly shaded screening pool 15 at well 15 at row A, column 15. The intersection occurs where the diagonal portion of one screening pool overlaps with the column portion of another screening pool. In other words, the intersection is the well in common with the two screening pools.
  • intersection shown in FIG. 7 occurs at the top of the diagonal and column.
  • the intersection first mentioned above for well 314 occurs at the bottom of the diagonal and column.
  • Other intersections occur between the two throughout the logical two-dimensional array in FIG. 7 so that the pooled substances of every well appear in two different screening pools. When screening occurs, it should identify the presence of an item of interest in each of two different screening pools.
  • the intersection of the two pools can be used to determine the coordinate locating the well containing the substance, such as a biological material, associated with the item of interest, such as a nucleotide sequence.
  • Screening pool 16 includes column 16 and the diagonal that begins with well 306 at row M, column 18 and extends up to well 374 at row B, column 29. However, the diagonal does not end at column 29 and wraps to column 1 as though it were present next to column 29 to include well 1 at row A, column 1. Thus, well 1 is the intersection of screening pool 16 and screening pool 1.
  • wells 373-384 could be positioned elsewhere, such as columns 13-24 of row N.
  • the diagonal of screening pool 15 that otherwise would have ended at well 373 would instead wrap to column 1 and include well 1. Accordingly, with the absence of column 29 from the logical array, screening pool 1 and screening pool 15 would intersect twice. One intersection is at well 1 and the other is at well 15. Consequently, a nonspecific deconvolution would exist and it would be impossible to determine, without further testing, whether the item of interest is in well 1 or well 15.
  • the positions of substances in only 378 of the 384 wells could be uniquely identified.
  • Column 30 may be added to the array so that the number of columns is greater than two times the number of rows plus one.
  • One way to add column 30 and still keep 14 rows includes duplicating wells 301 to 312 of row M (or other wells) in column 30. Of course, the duplication would create redundant testing.
  • Another way to add column 30 is to move wells 313 to 324 from row N to a new column 30. No duplication would exist, but the total number of tests to resolve the location of all 384 substances would increase from 29 to 30.
  • Another variation in the embodiments includes the one-dimensional array having bi-diagonal pools from a plurality of wells that do not have row or column identifiers in common and another equal number of wells having a row identifier in common with the plurality of wells.
  • such an embodiment also fits within the general criteria of screening pools including wells having a row identifier in common with one other well based on a pooling design that provides the pooled substances in two different screening pools.
  • FIG. 8 shows a grid with 29 columns and 384 wells as for FIG. 7, but includes bi-diagonal pools as described above. The embodiment of FIG. 8 further specifies that the other equal number of wells do not have row or column identifiers in common among themselves.
  • pseudo-column pools discussed above also apply to the bi-diagonal pools of FIG. 8. Although the application of the bi-diagonal pools is easily understood from their graphical depiction, other pooling designs are conceivable that provide some of the same benefits and fit within the general criteria for reducing a two-dimensional array to a one-dimensional array. Other benefits possibly unique to the pseudo-column pool and bi-diagonal pool embodiment include efficiency in robotic manipulation due to using contiguous wells for each pool. Reduction in robotic or manual manipulation during testing or sequencing reduces the significant costs of DNA sequencing library construction.
  • FIGS. 13 and 14 show one example of another pooling design that includes similar benefits to those of the pseudo-column pool and bi-diagonal pool embodiments.
  • the additional embodiment reduces a part of the two-dimensional array to a one-dimensional array and the screening pools from another part of the two- dimensional array form screening pools in a second dimension.
  • the number of screening pools is at least two times the number of rows plus one.
  • By forming one or more pools, such as row pools, to include the remainder of wells in the array well positions for substances might still be uniquely identified in the reduced number of screening pools enabled by complete reduction to one-dimension.
  • the row pools still have a row identifier in common with one other well based on a pooling design that provides the pooled substances in two different screening pools.
  • Lightly shaded screening pool 1 (column 1 ) in FIG. 13 includes both column and diagonal portions as for FIG. 7, but the diagonal is shorter in comparison.
  • the part of screening pool 1 in rows L to P does not have another equal number of wells providing a row identifier in common therewith. Accordingly, no intersections occur in rows L to P for screening pools 1 -24 designed as shown in FIG. 13. Even so, intersections occur in rows A to K as for the embodiment of FIG. 7. One such intersection is shown in FIG. 13 between screening pools 1 and 14 (column 14) at well 1.
  • row pools L to P designed as shown for row pool L in FIG. 14, intersect with the part of screening pools 1 -24 in rows L to P that does not have another equal number of wells in screening pools 1-24 with a row identifier in common.
  • screening pool 1 and row pool L intersect at well 265.
  • FIGS. 13 and 14 show a pooling design that provides the pooled substances in two different screening pools and allows determining the coordinates locating the well positions for all 384 wells of the array.
  • a three-dimensional array may also be reduced to a one-dimensional array using the principles described herein.
  • an additional diagonal or column might be added to the pools for FIGS. 7 and 8 or other two-dimensional arrays herein.
  • the additional diagonal or column of a screening pool may extend up out of the page, so to speak, into a third dimension.
  • Table 17 The theoretical increase in the number of individuals that can be uniquely identified when a three-dimensional array is reduced to a one-dimensional array is shown in Table 17 compared to the number of unique individuals identified for a two-dimensional array.
  • FIGS. 9a and 9b show a grid representing five 384-well plates and use of the embodiments herein for a collection of segregated substances placed in respective wells logically arranged in a two-dimensional array extending across all five plates. Only a portion of the logical array appears in FIG. 9a and it is continued to a second sheet in FIG. 9b.
  • the "columns" within the meaning of the embodiments extend along the left side of FIGS. 9a and 9b to form 64 columns using rows 1-16 of four plates.
  • the logical array includes 30 "rows" within the meaning of the embodiments that extend along the top side of FIGS. 9a and 9b and include columns 1 -24 of four plates and six additional columns of a fifth plate.
  • FIG. 10 shows a grid representing the fifth 384-well plate divided into four sections and demonstrates their incorporation into the logical array of FIGS. 9a and 9b, as described above.
  • the 61 or 91 experiments and 1 ,830 substances assume 24 columns and 13 rows on plate 4 are used for placing substances in wells. If more wells were used, then the location of some could not be uniquely identified with only the 61 screening pools of FIGS 9a and 9b. If 63 screening pools were used, then all 1920 substances of all five plates could be uniquely identified. With 64 screening pools used, then 1 more screening pool than needed is used but the ease of pooling may justify the extra screening pool.
  • FIG. 1 1 shows a grid representing twelve 384-well plates and use of the embodiments herein for a collection of segregated substances placed in respective wells logically arranged in a two-dimensional array extending across all 12 plates.
  • the "columns" within the meaning of the embodiments extend along the top of FIG. 11 to form 96 columns using rows 1 -16 of six plates.
  • the logical array includes 48 "rows" within the meaning of the embodiments that extend along the side of FIG. 1 1 and include columns 1 -24 of two plates. Consequently, the logical array accomodates two plates by six plates.
  • FIG. 11 only includes
  • the 12 plates of FIG. 1 1 may include 4,608 segregated substances. Using 96 experiments, the embodiment of FIG. 11 may be used to screen all of such substances placed in respective wells.
  • the logical array includes 96 pools (96 columns) instead of 97 pools, pool 1 and pool 49 overlap at two wells in the same manner as discussed above regarding FIG. 7 for the circumstance where 28 screening pools are used instead of 29. That is, the diagonal of screening pool 49 (column 49) wraps around the array past column 96 to intersect with column 1. As a result, the substances of 48 wells (one of the columns) are not uniquely identified, but the data is still gathered on all of the individuals even though location is not known exactly for about 1 % of the 4,608 individuals.
  • FIG. 12 shows screening pool 1 (column 1 ) intersecting with screening pool 48 (column 49) at just one well. Consequently, out of 4,608 substances, one appearing in both screening pools 1 and 49 can be identified at a unique location using only 96 tests.
  • the three-dimensional plate, row, column, diagonal repooled design uses fewer experiments but, practically, the tests are repeated due to wide confidence intervals and the increased difficulty in deconvolution of three of more dimensions.
  • the three-dimensional design seeks to identify the presence of an item of interest three times. Given the uncertainty of three identifications occurring, in practice, tests are repeated at least twice to avoid false negatives. Accordingly, in practice, at least 68 tests or more are used. A more narrow confidence interval exists for identifying the presence of an item of interest two times, as in the two-dimensional embodiment, so less motivation exists for repeating tests.

Abstract

L'invention concerne un procédé d'identification de substance, qui comprend la combinaison de substances en quatre ou plus de quatre sous-gisements intermédiaires dans des puits d'une plaque de sous-gisement et le remembrement des sous-gisements intermédiaires en un nombre de gisements de balayage finaux sur un modèle de remembrement fournissant les substances remembrées dans au moins trois gisements de balayage finaux différents. Le modèle de remembrement détermine des coordonnées localisant des positions de puits pour les substances. Un autre procédé d'identification de substance comprend l'utilisation d'un réseau bidimensionnel de puits agencé en rangées et d'un nombre de colonnes qui est au moins 1,5 fois les rangées. Les substances dans les puits sont combinées en un nombre de gisements de balayage. Des gisements de balayage individuels comprennent des substances provenant de puits ayant un identificateur de rangée en commun avec un autre puits. Un modèle de remembrement fournit les substances remembrées dans deux gisements de balayage différents. Le modèle de remembrement détermine des coordonnées localisant des positions de puits pour les substances.
PCT/US2012/062629 2012-10-30 2012-10-30 Procédés d'identification de substance à l'aide de remembrement WO2014070149A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2012/062629 WO2014070149A2 (fr) 2012-10-30 2012-10-30 Procédés d'identification de substance à l'aide de remembrement

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2012/062629 WO2014070149A2 (fr) 2012-10-30 2012-10-30 Procédés d'identification de substance à l'aide de remembrement

Publications (2)

Publication Number Publication Date
WO2014070149A2 true WO2014070149A2 (fr) 2014-05-08
WO2014070149A3 WO2014070149A3 (fr) 2016-06-09

Family

ID=47178963

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/062629 WO2014070149A2 (fr) 2012-10-30 2012-10-30 Procédés d'identification de substance à l'aide de remembrement

Country Status (1)

Country Link
WO (1) WO2014070149A2 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780222A (en) 1995-04-10 1998-07-14 Alpha Therapeutic Corporation Method of PCR testing of pooled blood samples
US6126074A (en) 1998-01-28 2000-10-03 Symbol Technologies, Inc. Error correction in macro bar code symbols
US6477669B1 (en) 1997-07-15 2002-11-05 Comsat Corporation Method and apparatus for adaptive control of forward error correction codes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8301388B2 (en) * 2003-05-05 2012-10-30 Amplicon Express, Inc. Pool and superpool matrix coding and decoding designs and methods

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780222A (en) 1995-04-10 1998-07-14 Alpha Therapeutic Corporation Method of PCR testing of pooled blood samples
US6477669B1 (en) 1997-07-15 2002-11-05 Comsat Corporation Method and apparatus for adaptive control of forward error correction codes
US6126074A (en) 1998-01-28 2000-10-03 Symbol Technologies, Inc. Error correction in macro bar code symbols

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AUSUBEL: "Short Protocols in Molecular Biology", WILEY AND SONS
S. ASAKAWA ET AL.: "Human BAC Library: Construction and Rapid Screening", GENE, vol. 191, 1979, pages 69 - 79
SAMBROOK: "Molecular Cloning, A Laboratory Manual", COLD SPRING HARBOR PRESS

Also Published As

Publication number Publication date
WO2014070149A3 (fr) 2016-06-09

Similar Documents

Publication Publication Date Title
Dunlap et al. Enabling a community to dissect an organism: overview of the Neurospora functional genomics project
Bouchez et al. Functional genomics in plants
Sprinzak et al. Correlated sequence-signatures as markers of protein-protein interaction
Alba et al. ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development
Twyman Principles of proteomics
Sato et al. Lotus japonicus as a platform for legume research
Ru et al. PhD7Faster: predicting clones propagating faster from the Ph. D.-7 phage display peptide library
Wilson‐Sánchez et al. Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants
Concia et al. Genome-wide analysis of the Arabidopsis replication timing program
US10155941B2 (en) High throughput yeast two-hybrid screening method and reagent kit
Reymond DNA microarrays and plant defence
Menon et al. Bioinformatics tools and methods to analyze single-cell RNA sequencing data
Suetsugu et al. Mycorrhizal communities of two closely related species, Pyrola subaphylla and P. japonica, with contrasting degrees of mycoheterotrophy in a sympatric habitat
Galbraith et al. Applications of microarrays for crop improvement: here, there, and everywhere
Urban et al. Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions
US20140121119A1 (en) Substance Identification Methods Using Pooling
US8301388B2 (en) Pool and superpool matrix coding and decoding designs and methods
WO2014070149A2 (fr) Procédés d'identification de substance à l'aide de remembrement
WO2001020998A1 (fr) Identification de medicaments au moyen d'un profilage de l'expression genique
AU2004234996B2 (en) Array having substances fixed on support arranged with chromosomal order or sequence position information added thereto, process for producing the same, analytical system using the array and use of these
US20030032014A1 (en) Colony array-based cDNA library normalization by hybridizations of complex RNA probes and gene specific probes
Willins et al. Genomics strategies for antifungal drug discovery-From gene discovery to compound screening
Schweizer et al. Both binding strength and evolutionary accessibility affect the population frequency of transcription factor binding sequences in Arabidopsis thaliana
Cameron et al. Genomic resources for the study of sea urchin development
Yardeni et al. Taxon‐specific or universal? Using target capture to study the evolutionary history of a rapid radiation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12787278

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12787278

Country of ref document: EP

Kind code of ref document: A2