WO2000023622A1 - Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique - Google Patents

Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique Download PDF

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Publication number
WO2000023622A1
WO2000023622A1 PCT/US1999/023906 US9923906W WO0023622A1 WO 2000023622 A1 WO2000023622 A1 WO 2000023622A1 US 9923906 W US9923906 W US 9923906W WO 0023622 A1 WO0023622 A1 WO 0023622A1
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Prior art keywords
label
cdna
library
dna
peptide
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PCT/US1999/023906
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English (en)
Inventor
Francois J.-M. Iris
Jean-Louis Pourny
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Valigene Corporation
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Priority to KR1020017004579A priority Critical patent/KR20010102909A/ko
Priority to CA002344625A priority patent/CA2344625A1/fr
Priority to JP2000577329A priority patent/JP2002527118A/ja
Priority to AU11130/00A priority patent/AU1113000A/en
Priority to EP99954899A priority patent/EP1121470A1/fr
Publication of WO2000023622A1 publication Critical patent/WO2000023622A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the present invention relates generally to methods of labeling, sorting, comparing and isolating populations of nucleic acids. More particularly, the present invention relates to a method for sorting and comparing complex populations of nucleic acid, such as cDNA libraries. These complex populations of nucleic acid may be derived from cells or tissue types having variations in phenotype of potential clinical interest.
  • the 10 methods are referred to generally as NaliGene SM Peptide-Labeled Oligonucleotide methods, or NG-PLO SM , and involve the use of distinguishable and identifiable peptide tags linked to oligonucleotide primers to manipulate nucleic acids.
  • Burdick and Oakes (Diagnostic Kit and Method Using a Solid Phase Capture Means For Detecting Nucleic Acids, European Patent Publication No. EP 0370 694 A2, Date of publication May 30, 1990) disclose the use of oligonucleotide primers, labeled with a label, with specific nucleic acid sequences which are complementary to a predetermined ° sequence of interest. This method is limited to the identification of a known sequence within a given sample and each pair of primers must correspond to a single predetermined PCR product.
  • This invention provides methods of labeling, sorting, comparing and screening multiple, complex populations of nucleic acids. These populations may be cDNA
  • ⁇ J_Q libraries constructed from phenotypically distinguishable cell or tissue types of interest. The method is extremely flexible and is adaptable to perform numerous complex sorting and comparing tasks.
  • the methods of this invention may also be used to increase and supplement the analytical powers of other techniques of manipulating complex cDNA population.
  • 15 major advantage of the methods of this invention is the ability to screen multiple populations of cDNAs derived, for example, from different tissues belonging to the same individual or to phenotypically different cell types present concurrently within a given tissue sample.
  • the invention employs oligonucleotide primers having distinguishable and identifiable peptide tags, which primers can be used to prime PCR reactions from vector sequences. Using such oligonucleotide primers, inserts from any given cDNA library can be labeled with library- specific peptide tags.
  • the distinguishable tags serve to identify the library-of-origin of any
  • the distinguishable tags can be used to selectively sort and isolate inserts based on their library of origin regardless of the complexity of the mixture of products. For example, one can use a chromatography matrix having an antibody specific to one of the distinguishable peptide tags. Such a matrix can trap or retain fragments, both single and double-stranded, which bear the specific peptide tag. Fragments which do not
  • the methods of the invention make use of polymerase chain reaction (PCR) primarily to linearize all inserts within a given cDNA library and to affix a distinguishable and identifiable peptide label to all inserts from a particular cDNA library so as to indicate their library-of-origin.
  • PCR can also be used at various stages to amplify complex mixtures of products.
  • Nucleic acid sample populations may be derived from many different sources. Such sources may include different phenotypes present concurrently within a given tissue sample or different tissues belonging to the same individual. One phenotype may, but does not need to be, "healthy" and another typical of a disease state.
  • the methods of the invention allow identification of genes that are specifically expressed in association with each phenotype as well as a comparison of genes which are expressed independently of the phenotype or are shared by some phenotypes but not all.
  • this invention provides a method comprising the following steps: (a) labeling DNA from each of a plurality of cDNA libraries using PCR with oligonucleotide primers having a label unique to each library; (b) contacting DNA labeled in step (a) with a first said label with DNA labeled in step (a) with a different said label and (c) sorting DNA contacted in step (b) using one or more molecules, each molecule being capable of binding the label unique to each library.
  • This invention further provides in the first embodiment additional methods wherein the label unique to each library is a 5'-peptide label.
  • This invention further provides in the first embodiment an additional method wherein the label unique to each library is biotin.
  • This invention further provides in the first embodiment additional methods wherein the one or more molecules is an antibody.
  • This invention further provides in the first embodiment, methods wherein the oligonucleotide primers prime PCR from vector sequences common to the nucleic acids within a particular library (thus a different one such primer is used for each library) or the oligonucleotide primer primes PCR from vector sequences common to the plurality of cDNA libraries (thus the same oligonucleotide primer is used for priming PCR for the entire plurality).
  • This invention further provides in the first embodiment a method of sorting which comprises (d) denaturing hybrid DNA strands resulting from step (b); (e) contacting single strands denatured in (d) with single strand binding protein to prevent strand reannealing; and (f) contacting the single strand binding protein coated single strands formed in (e) with one or more molecules, each molecules being capable of binding one of the labels unique to each library. 5
  • This invention further provides in the first embodiment a method wherein at least one of the one or more molecules in (e) is an antibody.
  • This invention provides in a second embodiment a method of cDNA library comparison comprising: (a) labeling DNA from a first cDNA population by PCR using oligonucleotide primers which have a first 5'-peptide label; (b) labeling DNA from a second 0 cDNA population by PCR using oligonucleotide primers having a second 5'-peptide label; (c) contacting DNA labeled in step (a) with DNA labeled in step (b) under conditions such that hybridization can occur and (d) separating DNA having the first and the second 5' peptide labels from DNA having only the first or the second 5' peptide label.
  • This invention further provides in the second embodiment additional 5 methods wherein the first cDNA population is from one or more cells or an organism subjected to a first condition and the second cDNA population is from one or more cells or an organism of the same type not subjected to said first condition.
  • This invention further provides in the second embodiment additional methods wherein the first cDNA population is from one or more cells or an organism o subjected to a first condition and the second cDNA population is from one or more cells or an organism of the same type subjected to a second condition.
  • This invention further provides in the second embodiment additional methods wherein the first and second cDNA populations are from cells or organisms that differ phenotypically. 5 This invention further provides in the second embodiment additional methods wherein the nucleotide sequences of the oligonucleotide primer pair having the first 5'-peptide label and the nucleotide sequences of the oligonucleotide primer pair having the second 5'-peptide label are the same.
  • this invention provides a method of monitoring gene 0 expression comprising: (a) contacting mRNA from a cell with an RNA-dependent DNA polymerase and a 5'-dephosphorylated target-specific primer (i.e., specific to the gene of which it is desired to monitor expression); (b) contacting any DNA:RNA hybrids synthesized in step (a) with a nuclease to remove single-stranded RNA extensions; (c) after step (b) ligating the DNA:RNA hybrids molecules to a partly double-stranded phosphorylated second primer (e.g., a primer that is not target specific); (d) labeling c products ligated in step (c) by PCR with a first primer complementary to the target-specific primer used in step (a), said first primer being labeled with a first label and a second primer complementary to one strand of the double-stranded phosphorylated second primer in (e), said second primer being labeled with a second label that is distinguishable from the
  • This invention further provides in the third embodiment an additional method wherein the nuclease is mung-bean nuclease. 5 This invention further provides in the third embodiment an additional method wherein the partly double-stranded phosphorylated second primer is an Ml 3 forward sequencing primer.
  • This invention further provides in the third embodiment an additional method wherein the first label is a peptide label. o This invention further provides in the third embodiment additional methods wherein at least one of the one or more molecules in step (e) is an antibody.
  • a fourth embodiment provides a method of identification of cDNA inserts represented in a first cDNA library and not represented in a plurality of other cDNA libraries comprising: (a) labeling DNA inserts from each cDNA library by polymerase chain reaction using oligonucleotide primers having a label unique to each library; (b) hybridizing DNA labeled in step (a); (c) contacting DNA hybridized in step (b) with a plurality of 0 immobilized antibodies capable of recognizing the label unique to each of the plurality of other cDNA libraries but not the label unique to the first cDNA library; and (d) recovering DNA which is not bound by the plurality of immobilized antibodies.
  • This invention further provides in the fourth embodiment additional methods wherein the DNA hybridized from each of the plurality of other cDNA libraries is in excess relative to the first cDNA library. Furthermore, additional methods are provided which c employ from a 2-fold to a 100-fold excess, from a 2.5-fold to a 10-fold excess and wherein the excess is a 3-fold excess.
  • This invention further provides in the fourth embodiment additional methods wherein the label unique to each library is a peptide label. Furthermore, methods are provided wherein the peptide label is 3 to 12 amino acid residues. Furthermore, methods Q are provided wherein the label is a thermophilic protein label.
  • This invention further provides in the fourth embodiment additional methods wherein one of the plurality of antibodies in step (c) is immobilized on a separate affinity column. Furthermore, methods are provided wherein the separate affinity columns are physically linked in series in any order. 5 This invention further provides in the fourth embodiment additional methods wherein the column flow-through is applied to the separate, physically-linked affinity columns one or more times. Furthermore, a method is provided wherein the column flow- through is applied to the separate, physically-linked affinity columns three times.
  • This invention further provides in the fourth embodiment a method wherein o the DNA retained by the antibody specific for the label unique to the first cDNA library is recovered and cloned.
  • this invention provides a method of identification of cDNA inserts represented in a first cDNA library and in a second cDNA library, and not represented in a plurality of other cDNA libraries, comprising: (a) labeling DNA from each 5 cDNA library by PCR using oligonucleotide primers having a label unique to each library;
  • step (b) hybridizing DNA labeled in step (a); (c) contacting DNA hybridized in step (b) with a plurality of immobilized antibodies capable of recognizing the label unique to each of the plurality of other cDNA libraries but not the label unique to the first cDNA library or the second cDNA library; and (d) recovering DNA which is not bound by the plurality of 0 immobilized antibodies.
  • This invention further provides in the fifth embodiment methods wherein DNA hybridized from each of the plurality of other cDNA libraries is in excess relative to the first and second cDNA libraries. Furthermore methods are provided wherein the excess is from 2-fold to a 100-fold excess, wherein the excess is from 2.5 fold to a 10-fold excess and wherein the excess is a 3-fold excess, c
  • the label unique to each library is a peptide label. Furthermore methods are provided wherein the peptide label is from 3 to 12 amino acid residues. Furthermore, methods are provided wherein the label unique to each library is a thermophilic protein label.
  • This invention further provides in the fifth embodiment methods wherein Q each of the plurality of antibodies in step (c) is immobilized on a separate affinity column.
  • This invention further provides in the fifth embodiment a method wherein the column flow-through is applied to the separate, physically-linked affinity columns one 5 or more times. Further a method is provided wherein the column flow-through is applied to the separate, physically-linked affinity columns three times.
  • This invention further provides in the fifth embodiment methods wherein DNA recovered in step (d) is further contacted with an antibody specific for the label unique to the first cDNA library or the label unique to the second cDNA library so as to concentrate o cDNA fragments specific to the first cDNA library and the second cDNA library.
  • methods are provided wherein the concentrated cDNA fragments specific to the first cDNA library and the second cDNA library are recovered and cloned.
  • methods are provided wherein the concentrated cDNA fragments specific to the first cDNA library and the second cDNA library are separated.
  • a method is provided 5 whereby the separation is carried out by denaturation, coating with single-strand binding protein and contacting with an antibody specific for the label unique to the first cDNA library or the second cDNA library.
  • this invention provides methods for matrix analysis of a plurality of cDNA libraries comprising: (a) labeling cDNA inserts from each of the 0 plurality of libraries with a distinguishable label; (b) hybridizing cDNA inserts labeled in step (a); (c) contacting cDNA inserts hybridized in step (b) with an affinity column capable of binding a distinguishable label; and (d) eluting the affinity column.
  • the distinguishable label is a peptide label
  • the step of labeling comprises priming PCR from cDNA library vector sequences by use of an oligonucleotide primer pair 5 having said peptide label attached to the 5 ends of said primer pair.
  • This invention further provides in the sixth embodiment a method wherein the labeled cDNA fragments from each library are hybridized in equal proportions.
  • This invention further provides in the sixth embodiment methods wherein the affinity column capable of binding a distinguishable label is an antibody affinity Q column. Furthermore a method is provided wherein the antibody- affinity column is eluted with a pH gradient.
  • This invention further provides in the sixth embodiment a method wherein eluted DNA is denatured to separate strands originating from two different libraries. Furthermore, a method is provided wherein the denatured strands are isolated by: (a) 5 coating with single-strand binding protein and (b) contacting with an affinity column capable of binding a distinguishable label.
  • the methods of this invention may also be used in another embodiment to construct subtracted cDNA libraries.
  • the sequences obtained from any of the above- described procedures may be used to remove a homologue from libraries known to share o such homologue or from any given unknown library.
  • the library to be subtracted is present as purified double-stranded clones.
  • This embodiment utilizes the ability of E. coli RecA protein to form stable triple-stranded structures between homologous sequences. Such triple-stranded structures are present as RecA coated single-stranded filaments and double- stranded linear and circular duplexes.
  • the method of this embodiment comprises: 5 (a) amplifying and labeling sequences identified as being shared by different libraries by
  • FIG. 1 Schematic representation of the results obtained from Phase I, Phase II and Phase III, respectively, of the antibody affinity columns used for sorting labeled 5 cDNA fragments.
  • FIG. 2 Schematic representation of the cDNA fragments comprising the input and output of Phase IV of the sorting process.
  • RNA:DNA hybrid produced by cDNA first-strand synthesis, including the target-specific primer on the 5' end of the cDNA strand.
  • FIG. 4 Ligation of the partly double-stranded standard primer of the RNA:DNA hybrid, including partial ligation to RNA strand only. 5
  • the present invention provides methods referred to generally as NaliGene SM Peptide-Labeled Oligonucleotide methods (VG-PLO SM methods) for manipulating (e.g., labeling, sorting, isolating and/or screening) two or more complex populations of nucleic o acids.
  • nucleic acids may be derived from a variety of sources, typically cD ⁇ A libraries representing different phenotypes.
  • the cD ⁇ A libraries used may represent phenotypes present (i) concurrently within a given tissue (e.g. normal and cancerous portions of a biopsy specimen) (ii) within different tissues belonging to the same or different individuals, (iii) among different cell lines, or (iv) within the same cell line 5 subjected to one or more different treatments.
  • PCR polymerase chain reaction
  • oligonucleotide primers to which peptide labels are linked are used to prime PCR reactions from vector sequences of cD ⁇ A libraries.
  • peptide labels and antibodies for binding said labels are used, in combination with a 5 suitable binding partner.
  • any label can be used, in combination with a 5 suitable binding partner. Examples of such labels and binding partners include, but are not limited to, digoxigenin-antidigoxigenin, biotin-streptavidin, ligand (e.g. hormone)-receptor and carbohydrate-lectin combinations.
  • the complexity of nucleic acid populations or mixtures will be understood by one of ordinary skill in the art to refer generally to the number of Q distinguishable clones in any given cDNA library or mixture of libraries.
  • the complexity of nucleic acids analyzed by the methods of the invention may vary over a very broad range. Generally, there is no upper or lower limit on the complexity of a population or mixture to be analyzed. For example, in one embodiment the complexity of a population or mixture may be from 10 to 10,000,000. Further, the complexity may be from 100 to 1,000,000. 5 Still further, the complexity may be from 500 to 500,000. In a preferred embodiment, the complexity of a mixture analyzed is about ( ⁇ 20%) 150,000.
  • the mixture of complexity ( ⁇ 20%) 150,000 comprises five libraries, each library having a complexity of about 30,000.
  • the complexity of the population being analyzed is at least 10 3 , 10 4 , 10 5 , or 10 6 .
  • the methodology of the invention utilizes library-specific labels linked to primer pairs capable of recognizing vector sequences of the vector used to construct the library. In this way, all nucleic acid fragments generated by PCR amplification with such primers have identical vector sequences at their 5' and 3' ends, yet also have a distinguishable label indicating the library-of-origin.
  • Described below 5 are methods for: sorting cDNA fragments to isolate those distinguishable to a single cDNA library; sorting cDNA fragments to isolate those common to two libraries; sorting cDNA fragments to isolate those common to multiple but not all libraries; and sorting cDNA fragments to isolate fragments shared only by two libraries out of all libraries analyzed.
  • set forth below are methods to construct subtraction libraries to monitor gene 0 expression events, and to isolate full-length transcripts of partial length sequences, such as expressed sequence tags.
  • vector-specific primers e.g., a peptide tag that comprises an epitope
  • the nucleotide sequences of a vector-specific primer pair can be identical among libraries.
  • each label (tag) is specific to a library-of-origin.
  • all fragments produced have (a) identical nucleotide sequences at their 5' and 3' ends corresponding to the vector-specific primer sequences and (b) a label indicating the library-of-origin.
  • the labeled inserts are then purified, e.g., by exclusion chromatography, to remove all reaction components, including excess peptide-labeled primers.
  • These purified, tagged PCR products are then combined, heat-denatured and allowed to reanneal.
  • the conditions under which renaturation and hybridization are carried out can vary.
  • the combined, heat-denatured PCR products are maintained together at 98 °C for ten (10) minutes.
  • the solution is then allowed to cool from 98 °C to 85 °C over a period of five (5) minutes.
  • the temperature of the mixture is maintained at 85 ° C for ten (10) minutes and then cooled to 65 ° C over a period of fifteen (15) minutes.
  • the solution is then maintained at a temperature of 65 °C for a further time period of fifteen (15) minutes. At this point, the reannealing process is considered complete.
  • the quantity of each PCR reaction product used in the hybidization reaction can vary. Since isolation of cDNA fragments specific to one library is desired, the other libraries are used in excess (i.e., as a "mop"). Specifically, where one wishes to isolate fragments present in library A but not in B, C, D and E, one uses excess B, C, D and
  • the amount of excess B, C, D, and E used will determine the efficiency of removal.
  • 3-fold excess of B, C, D and E is used.
  • from 2-fold to 100-fold excess B, C, D and E is used.
  • from 2.5 to 10-fold excess B, C, D and E is used. Since the function of using excess B, C, D and E is to act as a "mop" for removal of homologous cDNA fragments from the library-of- interest (in this example, library A), there is no restriction on the upper limit of the excess that can be used. However, a 3-fold excess will efficiently remove the fragments from library A that will form hybrids with B, C, D and E without being wasteful.
  • Aliquots of the reannealed mixture are then contacted with one or more solid 5 phases, e.g., by passage through separate chromatography columns, having a binding partner, preferably antibodies (Abs), specific for tags B, C, D and E, respectively.
  • a binding partner preferably antibodies (Abs)
  • a single solid phase e.g., column, having antibodies specific for all four tags may be used.
  • Methods of making Ab affinity columns are well known.
  • agarose beads e.g., SepharoseTM or Sepharose CL, Pharmacia
  • the beads are washed with dioxane in water and incubated at room temperature with carbonyldumidazole.
  • an antibody that specifically binds to a short peptide of from 6 to 12 amino acids that is used as the label/tag.
  • peptides of any length can be used as "tags" if the chosen peptide is known to spontaneously renature following heat denaturation (e.g. thermophilic proteins).
  • the antibody releases the retained 5 peptide label when placed in a weakly acidic solution (e.g., pH 5.5). Elution of antibody- affinity columns on the basis of pH gradient is another preferred embodiment of this invention.
  • the flow-through can be applied to each B, C, D and E column or other solid phase one or more times. Multiple times is preferred. After several cycles, all or most of 0 the fragments bearing B, C, D or E tags, on either single or double-strands, will be trapped.
  • the flow-through is applied to the column three times.
  • the flow-through will generally contain single and double-stranded fragments bearing the A tag only.
  • the temperature for running the antibody affinity columns may vary. In a 5 preferred embodiment the temperature is from 4°C to 50°C. In a most preferred embodiment, the antibody-affinity columns are water-jacketed and maintained at a temperature of 37°C.
  • the B, C, D and E columns are next washed with a low-salt (e.g., 50mM NaCl) buffer (e.g., phosphate buffer).
  • a low-salt e.g., 50mM NaCl
  • the Q pooled flow-through and washes from the B, C, D, and E columns are then passed through a column containing an A-specific antibody only.
  • the trapped A-tagged fragments can then be eluted, precipitated and amplified using PCR with unlabeled, vector-specific primers.
  • the A-tagged fragments are amplified using 20 cycles of PCR and cloned for analysis. These cloned fragments are highly enriched for fragments specific to 5 library A (i.e., fragments not found in library B, C, D, or E).
  • any method known in the art may be used to elute peptide-labeled fragments from antibody affinity columns used in the various embodiments of this invention.
  • columns are eluted by changing pH (pH gradient).
  • the antibodies, or other solid phase, chosen will o release the peptide label in a weakly acidic pH (e.g., 5.5).
  • the same series of steps can be used to isolate cDNA fragments tagged with B, C, D or E peptides by changing the "mop".
  • To isolate the fragments labeled with B one would start with an A, C, D and E multi-antibody affinity column to retain everything but B-labeled fragments. The pooled flow-through and washes of this column 5 would then be passed over a column containing only a B-specific antibody.
  • the same approach can be used to isolate fragments specific to the C, D and E libraries.
  • the material trapped in the first column will contain all fragments tagged with B, C, D and E labels. This will include hybrids containing one A-tagged strand. This 0 material can also be eluted and further sorted using other embodiments of the invention, or as otherwise desired by the practitioner.
  • the methods of this invention are used to isolate cDNA fragments that are common to, (i.e., that will form hybrids with) cDNA fragments from one . . . or more of the other libraries. For example, if one starts with a plurality of cDNA libraries
  • this embodiment allows the isolation of transcripts common to the
  • a and C libraries (or to any other two specified libraries).
  • isolation of cDNA fragments from the A library which form hybrid duplexes with cDNA fragments from the C library is described.
  • the inserts from each of a plurality of cDNA libraries are linearized and tagged by PCR with a distinguishable library-specific label attached to vector-specific primers. Accordingly, as above, all fragments produced have identical nucleotide sequences at their 5' and 3' ends corresponding to the vector-specific primer sequences, and a distinguishable label indicating the library-of-origin.
  • the labeled PCR products are then, purified by exclusion chromatography to remove all reaction components, including excess labeled primers.
  • the purified, labeled PCR products are then combined, heat-denatured and allowed to reanneal.
  • the quantity of each PCR reaction product used in the hybridization can vary.
  • one would use an excess of libraries B, D and E.
  • the purpose of this excess is to act as a "mop" as in the previous approach.
  • a 3-fold excess will efficiently remove cDNA fragments from both the A and C libraries that will form hybrids with any fragments from B, D and E libraries without being wasteful.
  • a 3-fold excess is the preferred embodiment.
  • the reannealed mixture is then passed through a chromatography column containing antibodies specific to the labels on all the libraries except the two of interest.
  • the column would contain antibodies to labels on libraries B, D and E.
  • the flow-through of this multi-antibody column can be applied one or more times. However, multiple times is preferred since each pass-through will increase the percentage of B, D or 5 E-labeled fragments which will be retained in the column and therefore removed from the flow-through. After several cycles, all or most of the fragments which bear the B, D or E label will be removed and the flow-through will contain only fragments bearing the A or C label. These fragments will consist of A: A and C:C duplexes and, in addition, may contain A:C hybrids (i.e., the fragments-of-interest). These A:C hybrids contain cDNA fragments
  • the A:C hybrids are the product of interest.
  • the mixture containing A: A and C:C duplexes and A:C hybrids is next passed through an antibody affinity column with immobilized anti-A label antibody. This column will retain all or most fragments which
  • A-label This will include A: A duplexes and the A:C hybrids of interest.
  • the flow-through may be applied to this single-antibody column one or more times. The amount of A-labeled fragments retained will be increased with each pass. In a preferred embodiment, the flow-through is passed through the column three times.
  • the recovered material consists of A: A duplexes and A:C hybrids. These double-stranded fragments are then heat denatured.
  • the resulting single strands are cooled rapidly to prevent renaturation.
  • this can be accomplished by rapidly cooling the heat-denatured material on a bath of dry-ice and methanol.
  • Single-strand binding 25 protein (SSB) is added to the frozen mixture. This protein will stabilize single DNA strands by coating them, thereby preventing renaturation.
  • the SSB is then added in excess to the frozen mixture of denatured single-strands. This frozen mixture is then warmed to allow the SSB to enter the solution and contact the single strands.
  • the mixture is heated from the temperature of the dry-ice/methanol bath to
  • the SSB will coat and stabilize the single DNA strands and will prevent reformation of hybrids and the formation of secondary structures.
  • the single strands of DNA consist of fragments from the A library that formed hybrids with other fragments from the A library, and fragments from the C library that formed hybrids with fragments from the A library.
  • the C library fragments present at c this stage will be limited to those fragments that were able to hybridize with A library fragments and were thus retained on the anti-A antibody column. In addition, these C library fragments did not hybridize with and thus were not removed by the excess of B, D and E fragments used as the "mop".
  • these C library fragments are represented in the A library but not in the B, D, or E library.
  • a further step is now employed to separate the SSB- coated A and C single strands.
  • This step consists of passing the SSB-coated single-strands through an antibody-affinity column.
  • This column may contain either immobilized anti-A antibody or immobilized anti-C antibody. If the anti-A column is used, then A-labeled fragments will be retained by the column and the C tagged fragment will remain in the 5 flow-through and washes. If the anti-C antibody column is used, then the C-labeled fragments will be retained by the column and may be eluted from this column.
  • the A-labeled fragments and the C-labeled fragments can be recovered.
  • the recovered fragments are then extracted to remove SSB, and PCR amplified.
  • These C-labeled fragments may be cloned for further analysis or they can be used as pooled probes (i.e., o "subtraction probes") to remove their homologues from the original A or C libraries (see e.g., Section 5.4 below).
  • more than two libraries- of-interest may be designated. For example, if one is interested in fragments that may form hybrids between library A, B and C but not with libraries D and E, then the first step would 5 employ an excess of D and E PCR reaction products over those of A, B and C. In this embodiment the multiple antibody column would contain anti-D and anti-E antibody. The excess of D and E used would form the "mop" to remove cDNA fragments that formed hybrids with any fragments in the A, B, or C libraries.
  • the flow-through and washes of this multi-antibody column would contain A: A, B:B and C:C duplexes but would also contain 0 A:B, A:C and B:C hybrids if any had formed. These hybrids are the products-of-interest in this embodiment. If this material is now passed through an anti-A column, then A:B and A:C hybrids will be retained. An anti-B antibody column will retain any A:B and B:C hybrids, and an antibody column containing anti-C antibody will retain C:B and C:A hybrids. The material retained on these three columns may be eluted and isolated by the methods used above.
  • an array or matrix comparison is made among a plurality of cDNA libraries constructed and manipulated, in part, using variations of the procedures set forth above.
  • any cDNA fragments present in two of N libraries can be isolated, where N is the total number of libraries 5 subjected to the matrix analysis.
  • any cDNA fragments present in X of N libraries can be isolated, where X is the number of libraries in which the particular cDNA fragments isolated are found.
  • any cDNA fragment present in only two (or three, or X) of N libraries can be isolated.
  • the cDNA fragments common to any desired number (X) of any number (N) of libraries analyzed can be isolated, and whether or not these o fragments are exclusively shared among a subset of N libraries analyzed can be determined.
  • a major advantage of this embodiment is the absence of a necessity for having any knowledge of which genes (i.e. cDNA fragments) may or may not be represented in a given cDNA library before beginning the comparative analysis. Further, the degree or extent of homology (i.e. similarity) among cDNA fragments obtained from a 5 plurality of libraries also need not be known. Still further, one need not know whether a specific library-of-interest shares any similar cDNA inserts with any other libraries prior to beginning the analysis.
  • the cDNA inserts of each library are separately linearized and labeled by PCR with a label distinguishable to each library. Again, a 5'-peptide label is preferred. As above, the peptide label distinguishable to each library is attached to the 5' ends of an oligonucleotide primer pair used to prime PCR from library vector sequences.
  • each c labeled library is separately purified, e.g., by exclusion chromatography.
  • This step purifies the linearized, labeled inserts away from unwanted reaction components, such as excess peptide-labeled primers.
  • This purification can be performed by any of the standard methods well known in the art (e.g., PCR purification kit from Qiagen, Santa Clarita, California).
  • the purified and distinguishably labeled cDNA fragments from each of the Q five libraries are then mixed together, heat denatured and allowed to re-anneal. The relative proportions of material from each library mixed together in this reaction is determined by user discretion.
  • the reaction may or may not employ an excess of material from one or more libraries over another one or more libraries.
  • the labeled cDNA fragments from each library are mixed together in equal proportions. 5
  • this embodiment performs a comparative analysis of cDNA libraries based on hybridization and sorting of labeled cDNA inserts.
  • the analysis employs up to four stages or Phases of affinity columns, or any solid phase, capable of binding specific library labels.
  • any label known in the art suitable for labeling cDNA strands by PCR may be used.
  • any affinity column, or any solid phase, known in the art, capable of binding such labels may be used.
  • the affinity columns are antibody-affinity columns capable of binding peptide labels.
  • Phases employed in this embodiment are determined in part by the result desired by the user.
  • all products of 5 the comparative array created need not be analyzed and may be stored.
  • the analysis need only proceed through Phases I and II.
  • Phases I, II and III are 0 employed.
  • Phases I, II and IV are employed.
  • a library "Group” is defined as the eluent obtained from a Phase I column, as further set forth in the sections below. The following narrative describes the steps employed to achieve the results just described. As noted above, while described in terms of antibody affinity columns and peptide labels, other types of solid phases with other types of binding partners to other types 5 of label, can be used.
  • the re-annealed mixture of labeled cDNA fragments is applied sequentially to a series of antibody affinity columns, each column having an immobilized Q antibody capable of recognizing only one of the library labels.
  • the re- annealed mixture may be divided into aliquots and applied to each column individually.
  • five columns are used.
  • the order of application of the re- annealed mixture to the five individual columns can be any order.
  • the order can be A column, B column, C column, D column and E column, respectively.
  • the five columns are physically linked in series.
  • a through E were physically linked for application of the cDNA mixture and washes, they are dis-assembled prior to elution.
  • the material obtained from elution of each Phase I column defines a separate library Group, as follows:
  • Library Group A from A column - double-stranded A:A, A:B, A:C, A:D and A:E duplexes, and single-stranded A-labeled DNA
  • Library Group B from B column - double-stranded B:A, B:B, B:C, B:D and B:E duplexes, and single-stranded B-labeled DNA
  • the material-of-interest is duplex DNA formed from hybridization of strands originating in two different libraries. Where five libraries are used for the input mixture of Phase I, as in this example, twenty different duplexes-of-interest may be formed. For example, in column A, trapped library Group A duplexes-of-interest consist of A:B, A:C, A:D and A:E duplexes. In column B, trapped library Group B o duplexes-of-interest consist of B:A, B:C, B:D and B:E duplexes, etc. See FIG. 1, Phase I, for a complete listing of the array of products produced. Duplexes having identical labels on each strand and any single-stranded DNA trapped in Phase I columns is not generally of interest and is therefore not shown in FIG. 1.
  • the cDNA fragments (i.e. transcripts) shared between any two libraries-of- interest is isolated in Phase II.
  • the eluent from each of the five Phase I columns is rendered single-stranded prior to input over Phase II columns. This may be performed by any method known in the art.
  • single-strand binding protein 0 (SSB) is used.
  • SSB single-strand binding protein 0
  • the eluent from each of the five Phase I columns (Groups A through E in FIG. 1) is separately heat-denatured and rapidly cooled (e.g. dry-ice/methanol bath). An excess of SSB is added, and each SSB-plus-DNA mixture is then warmed.
  • the temperture is increased to and maintained at 37 °C for 10 to 15 minutes.
  • each series of Phase II columns is physically linked.
  • Each Phase II column contains a single immobilized antibody specific for one of the distinuishable peptide labels used to label the input cDNA libraries.
  • a Phase II series of columns may contain as many columns as the number ofcDNA libraries N being analyzed.
  • a Phase II series of columns contains N-l columns, where the omitted column corresponds to the library Group (e.g. for library Group A, the A column may be omitted).
  • each Phase II column captures single strands bearing one of the distinguishable peptide labels.
  • Each Phase II column is then separately eluted. In this way, an array of twenty groups of single-stranded cDNA fragments is isolated wherein each of the twenty groups contains fragments shared (i.e. hybridizable) between two libraries (see FIG. 1, Phase II).
  • cDNA fragments are isolated in single-stranded form: B column - cDNA fragments originating from the B library also present in the A library; C column - cDNA fragments originating from the C library also present in the A library; D column - cDNA fragments originating from the D library also present in the A library; and
  • E column - cDNA fragments originating from the E library also present in the A library.
  • the following cDNA fragments are isolated in single- stranded form:
  • a similar list of isolated cDNA fragments can be constructed for each series of Phase II columns, thereby completing the array (see FIG. 1, Phase II).
  • any fragments in the array created by the output of the Phase II columns may be cloned for further analysis as desired by the user.
  • Such fragments may also be used as "combination probes" for retrieval of corresponding double-stranded clones from an existing library using, for example, the RecA method detailed elsewhere herein.
  • These fragments are also used as input to Phase III and Phase IN for isolation of cD ⁇ As exclusively present in two or more desired libraries, either within or across library Groups, respectively, as further set forth below.
  • Phase III is used to isolate fragments shared exclusively between two or more designated members within a library Group. For example, Phase III allows isolation of fragments shared exclusively between libraries A and B, A and C, A and D, and A and E, within library Group A. Further, Phase III allows isolation of fragments shared exclusively between libraries B and A, B and C, B and D, and B and E, within library Group B. This pattern is equally applicable to library Groups C, D and E.
  • Phase III begins with single-stranded fragments eluted separately from each of a series of Phase II columns in a chosen library Group as described above. These fragments are first independently amplified by PCR and labeled with the relevant peptide label. For example, where library Group A is being subjected to a Phase III analysis, fragments eluted from the B column of Phase II are amplified using the B-specific peptide label, fragments eluted from the C column of Phase II are amplified using the C-specific peptide label, etc.
  • All the independent members belonging to a given library Group are 5 likewise amplified and labeled by PCR.
  • the amplified products are then mixed, denatured and allowed to re-anneal.
  • the reannealed Phase III input mixture is then divided into four aliquots. The number of aliquots needed depends on the number of separate labels in the mixture.
  • the A library Group is analyzed and the labels used during the PCR Q amplifying process are B, C, D and E.
  • the Phase III columns consist of four series of affinity columns, each series consisting of three single-antibody columns. Each of these four series of columns contain antibodies specific to three of the four labels used in the Phase III PCR amplification step.
  • the four series of affinity columns contain: 5 Series 1 - C, D and E antibodies;
  • Phase III Series 1 retains any cDNA fragments labeled with C, D and E, o allowing B-labeled duplexes and single strands to remain in the flow through.
  • these cDNA fragments are present in libraries A and B, but not in C, D or E. Therefore, cDNA fragments exclusively present in libraries A and B have been isolated.
  • Phase III Series 2 allows only C-labeled fragments to pass
  • Phase III Series 3 allows only D-labeled fragments to pass
  • Phase III Series 4 allows only E- 5 labeled fragments to pass.
  • cDNA fragments exclusively present in libraries A and C, A and D, and A and E, respectively, have been isolated.
  • each series column one uses one column less than the number of labels used in the amplifying step. Further, one uses enough series to cover all different combinations of columns.
  • the flow-through of each of the four Series of multi-antibody columns just described above is next passed through another antibody column. These columns each contain a single antibody which is specific for the labeled fragments allowed to pass in the Phase III Series columns. This step serves to concentrate the fragments, which otherwise might be difficult to recover from a large volume of flow- through and washes. The fragments retained by these four single-antibody columns are eluted and recovered. This material consists of concentrated cDNA fragments that are uniquely shared between two specific libraries. In this example, the fragments recovered are uniquely shared between libraries A and B, A and C, A and D, and A and E within library Group A.
  • the output from Phase II can be further analyzed in Phase IV to determine whether cDNA fragments shared between any two libraries-of-interest in the array are distinguishable across library Groups rather than within library Groups.
  • the Phase IV analysis thus complements the Phase III analysis by allowing one to ask essentially the same question using different input cDNA fragments.
  • the user thus benefits by comparing the results of a Phase III analysis with the results of a Phase IV analysis.
  • the input DNA for Phase IV analysis is obtained from the output of Phase II.
  • the labels attached in the PCR reactions prior to Phase IV analysis correspond to the library Group label and not to the original label of the fragment (see Box in FIG. 2).
  • the labels attached in the PCR prior to Phase IV analysis of Phase II products do not correspond to the library-of-origin of a particular fragment. Instead, the labels correspond to a library in which a given fragment has found a homolog (i.e. the library Group). In this way, an analysis similar to the Phase III analysis can be performed across library Groups. For example, to perform a Phase IV analysis across library Groups, all fragments originating from A library and recovered from a B group column in Phase II are labeled with B peptide label (see Box in FIG. 2 at "Tag-B").
  • each multi-antibody column contains three label-specific antibodies.
  • Phase IV analysis is performed for c fragments originating in A library
  • four Series of Phase IV columns contain:
  • Phase IV Series column may then be pooled and applied to a column containing a single antibody specific for the one label that remained untrapped.
  • the output of the Phase IV Series 1 column above would be pooled and passed over a column containing anti-B. Representative results for fragments originating in A library and for fragments originating 5 in B library are shown in FIG. 4 (see "output of Phase IV").
  • the material eluted from each single-antibody column in Phase IV consists of concentrated cDNA fragments shared exclusively by two libraries (i.e., not found in the other libraries of the analysis).
  • the methods of this invention can be used to 0 construct subtracted cDNA libraries, i.e., to remove similar clones from two or more cDNA libraries.
  • the method described herein takes advantage of the E. coli RecA protein's ability to form stable triple-stranded structures as recombination intermediates. RecA catalyzes a homologous pairing and strand exchange reaction during E. coli homologous recombination. During the first step of this reaction, RecA coats a single strand of DNA and initiates an exchange reaction between the single strand and a homologous region of 5 double-stranded DNA.
  • a three-stranded nucleoprotein intermediate is formed, which, in the absence of ATP, is surprisingly stable (see West, 1992, Annu. Rev. Biochem., 61:603- 640).
  • cDNA fragments-of-interest such as those shared by different libraries identified as described above, are amplified by PCR using peptide-tagged vector-specific Q primers. Thus a distinguishable peptide tag marks a given set of cDNA sequences. Such sets of fragments are used concurrently as "subtraction probes".
  • a subtraction probe set is purified by exclusion chromatography following PCR and heat-denatured. The cDNA mixture is then flash frozen (e.g., dry-ice/methanol).
  • RecA protein is added to the ice pellet along with non-hydrolyzable ATP (e.g., ATP ⁇ S).
  • ATP non-hydrolyzable ATP
  • the ice pellet is slowly 5 thawed. Low temperature and the ATP analog prevent the RecA-bound single-stranded DNA from renaturing so that the subtraction probe remains single stranded and becomes coated with RecA.
  • the library to be subtracted, in the form of purified double-stranded, circular DNA, is added to the thawed pellet such that the RecA-coated single strands are present in o large excess (20-50 fold).
  • the mixture is heated to 37°C.
  • the RecA-coated single strands scan the double-stranded cDNA library in search of homologous sequences, and pair with such sequences. Triple-stranded recombination intermediates are formed, although strand exchange will not occur due to the absence of a hydro lyzable form of ATP.
  • the triple- stranded structures formed from single-stranded DNA and homologous double-stranded 5 DNA are labeled with the specific peptide tag bound to the single strand.
  • triple- stranded, labeled structures can now be separated from unlabeled, double-stranded circular molecules by passing the solution through a peptide tag-specific antibody column. Most or all clones corresponding to the labeled fragments will be removed from the library if the RecA-coated single strands are present in large excess over the plasmid clones. 0
  • the method of this embodiment is independent of the original nature of the nucleic acid used to construct the library. It can therefore be used with DNA libraries made from cDNAs or genomic DNAs.
  • both single-stranded fragments and double- stranded library plasmids share identical extremities (i.e., 5' and 3' ends) over at least 10-15 bases, and the homologous fragments are at least 350 bp in length. If strong overall 5 homology is present, perfect identity between fragments is not required for RecA to form stable triple-stranded structures (see, e.g., Rao et al.,1995, Trends In Biological Science 20: 109-113).
  • the cloned inserts do not exceed 1-2 kilobase (kb) in length so that clones sharing only strong localized homologies with the subtraction probes are not selected.
  • oligonucleotides labeled with specific and identifiable peptide labels are used, but in this embodiment the targets (i.e. genes) to be monitored for expression are known. These targets may belong to an expression cascade, for example, if ⁇ -5 the objective is to define the mechanism of action or physiological effects of a particular drug treatment.
  • An alternative use for the methods of this embodiment is to monitor gene expression to define a phenotype based on the activation or repression of a specific phenotype-associated metabolic pathway. The advantage of this method is to provide a simple and rapid means to sort, separate and quantify the product (representing targets to be ° monitored for expression) based on the peptide label.
  • PCR reaction is carried out using unamplified cDNA from a first-strand synthesis reaction. For a fixed and limited number of PCR cycles (e.g., from about 5 to 20 cycles), the product of the reaction is
  • the methods of this embodiment will allow the direct monitoring of gene expression events, as well as the isolation of partial length transcripts, without the prior
  • this embodiment allows its use to analyze the response of a specific phenotype to a given stimulus or set of conditions.
  • this method provides a rapid and accurate means of directly determining the physiological 5 effects of any form of treatment which affects gene expression, such as treatment with steroid hormones. Since this is done directly by looking at mRNA production, it is not necessary to wait for the overt clinical effects to show themselves or the production of serological factors.
  • the method of this embodiment could therefore be used to make a rapid assessment of the probable effect of treatment, or to provide rapid and direct feed-back to Q allow therapeutic readjustments to be made to optimize outcome.
  • total RNA is extracted from the tissue sample using standard methodologies well known to those skilled in the art.
  • Total RNA is used for hybridization to target-specific probes.
  • Each of these probes consists of a synthetic oligonucleotide labeled with a specific peptide epitope or tag at the 5' end and a fluorophore 5 at the 3' end.
  • the single-stranded probes are mixed with the samples of total RNA under conditions allowing hybridization of the probes to their target mRNA molecules, if present.
  • the mix is treated with a single-strand specific DNase in order to destroy all non-hybridized excess probes or to effect a separation between the peptide tag and the fluorescence label on probes remaining single-stranded.
  • a single-strand specific DNase in order to destroy all non-hybridized excess probes or to effect a separation between the peptide tag and the fluorescence label on probes remaining single-stranded.
  • detectable labels may substitute for the fluorescence label.
  • the mixture is then exposed to a solid surface onto which the tag-specific antibodies or other binding partners have been arrayed (e.g. an ELISA plate) hence identifying the relative position of each target-specific probe.
  • This embodiment can also be used to isolate full- length forms of only partial-length transcripts.
  • Total RNA is extracted as previously and aliquots of the total 0 RNA are used for cDNA first-strand synthesis using target-specific, non-phosphorylated primers (see, FIG. 3).
  • the synthesis makes use of an RNA-dependent DNA polymerase (i.e., reverse transcriptase) which does not possess RNase-H activity (such as Moloney murine leukemia virus reverse transcriptase).
  • reverse transcriptase RNA-dependent DNA polymerase
  • Methods for doing this are well known to those 5 skilled in the art (Sambrook et al., 1989, Molecular Cloning A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press).
  • the result of this synthesis is DNA:RNA hybrids with a target-specific primer on the 5' end of the DNA strand. Any RNA extensions can now be removed to produce blunt ends by treatment with mung-bean nuclease, which cleaves single-strand mRNA extensions.
  • Q This reaction can be performed under standard conditions for the use of mung bean nuclease.
  • the DNA:RNA hybrids may be suspended in a mung bean nuclease Buffer consisting of 50 mM sodium acetate (pH 5.0 at 25 °C), 30 mM NaCl, 1 mM ZnSO 4 .
  • Mung bean nuclease in the amount of 1.0 unit per microgram of DNA:RNA hybrid is added and the mixture is incubated at 30 °C for thirty (30) minutes.
  • the enzymes 5 may then be inactivated by phenol/chloroform extraction or by addition of SDS to 0.01%.
  • the blunt-ended hybrids may be recovered by alcohol precipitation.
  • the sample is now purified by standard exclusion chromatography. After purification, the sample consists of the DNA:RNA hybrids together with the remainder of o the total RNA species initially present. The exclusion chromatography removes the small
  • RNA species such as tRNA
  • excess target-specific primer excess target-specific primer
  • a ligation reaction is carried out using DNA ligase from T 4 bacteriophage.
  • the ligase will catalyze the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini of DNA or RNA and will thus join the 3' end 5 of a double-stranded DNA fragment to the 5' terminus of a double-stranded DNA:RNA hybrid molecule.
  • the primer used is a partly double-stranded phosphorylated second primer (i.e. a primer that is not target-specific), for example, a Ml 3 "forward" sequencing primer (see FIG. 4).
  • Bacteriophage T 4 DNA ligase will fully ligate the primer only to the 0 phosphorylated end of the DNA:RNA hybrid. However, some of the primer molecules will also ligate to the 3' terminus of the RNA strand of the DNA:RNA hybrid. This will not affect the result because DNA polymerase enzyme in subsequent steps will not use RNA as a template and because no template is available in the 3' direction and so priming at this site will not result in elongation by de novo synthesis.
  • T 4 DNA ligase purified from E. coli may be obtained from New England 5 Biolabs (Waverly, Massachusetts). The reaction may be carried out in T 4 DNA Ligase Buffer which contains 50 mM Tris-HCl (pH 7.8), 10 mM MgCl 2 , 10 mM dithiothreitol, 1 mM ATP, 25 microgram per milliliter bovine serum albumin. In a preferred embodiment, the rection is carried out at 16°C for between four (4) and sixteen (16) hours. Engler, MJ. and Richardson, C.C. (1982) in The Enzymes (Boyer P.D., ed.) Vol. 5, p. 3, Academic Q Press, San Diego, CA.
  • the sample is again purified by exclusion chromatography and amplified by PCR.
  • the PCR reaction makes use of both a pep tide- tagged primer complementary to the target-specific primer previously used, and a biotinylated primer complementary to the partly double-stranded phosphorylated standard primer used in the ligation reaction.
  • this PCR reaction includes 50 nanograms of yeast RNA per 30 microliters of solution. The number of cycles in this PCR reaction can vary. In a preferred embodiment, 20 or fewer cycles is used.
  • the sample can be treated with RNase immediately following the ligation reaction and prior to PCR. This will destroy all RNA 0 strands, including single strands of total RNA and the RNA strands of the DNA:RNA hybrid molecule.
  • the sample can then be purified by exclusion chromatography, and PCR amplified and labeled as above. The amplified product is then purified by exclusion chromatography to remove all excess primers.
  • the amount of product produced by the PCR reaction can now be quantified 5 by a modification of an enzyme-linked immunoassay technique (ELISA).
  • the purified reaction mixture may be analyzed in microtiter wells coated with an antibody specific to the peptide label that was attached to the target-specific primer. Streptavidin-linked horseradish peroxidase can then be added to bind to the biotin moiety attached to the standard primer of the retained PCR products. A horseradish peroxidase substrate can then be added, and the reaction product quantified (see e.g. Sambrook et al, 1989, Molecular Cloning A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press at 18.75), indicating the amount of target mRNA present in the original sample.
  • ELISA enzyme-linked immunoassay technique
  • target-specific primers can be used in the first- strand synthesis reaction.
  • Identifiable and distinct peptide-labeled primers complementary to the target-specific primers can be used in the PCR reaction.
  • the primers involved are chosen to be compatible in terms of their melting temperatures (Tm's) and propensities for secondary structure formation.
  • the input phenotypes represented by cDNA libraries employed in the methods of this invention can be chosen as desired by one skilled in the art.
  • This co-pending application is incorporated herein by reference in its entirety.
  • PCR polymerase chain reaction
  • a source e.g., a tissue sample, a genomic or cDNA library.
  • Oligonucleotide primers representing known sequences can be used as primers in PCR.
  • PCR is typically carried out by use of a thermal cycler (e.g., from Perkin- Elmer Cetus) and a thermostable polymerase (e.g., Gene AmpTM brand of Taq polymerase).
  • the nucleic acid template to be amplified may include but is not limited to mRNA, cDNA or genomic DNA from any species.
  • the PCR amplification method is well known in the art (see, e.g., U.S. Patent Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al, 1988, Proc. Nat'l. Acad. Sci. U.S.A. 85, 7652-7656; Ochman et al., 1988, Genetics 120, 621-623; Loh et al, 1989, Science 243, 217-220).
  • nucleic acid source Any prokaryotic cell, eukaryotic cell, or virus, can serve as the nucleic acid source.
  • nucleic acid sequences may be obtained from the following sources: human, porcine, bovine, feline, avian, equine, canine, insect (e.g., Drosophila), invertebrate (e.g., C. elegans), plant, etc.
  • the DNA may be obtained by standard procedures known in the art (see, e.g., Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., 5 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II).
  • nucleic acid hybridization under low, moderate, or high stringency conditions include nucleic acid hybridization under low, moderate, or high stringency conditions (e.g., Northern and Southern blotting).
  • Methods for adjustment of hybridization stringency are well known in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; see, also, Ausubel et al., eds., in the Current Protocols in Molecular Biology 5 series of laboratory technique manuals, 1987-1994 Current Protocols, 1994-1997 John Wiley and Sons, Inc.; see, especially, Dyson, N.J., 1991, Immobilization of nucleic acids and hybridization analysis, In: Essential Molecular Biology: A Practical Approach, Vol.
  • Salt concentration, melting o temperature, the absence or presence of denaturants, and the type and length of nucleic acid to be hybridized are some of the variables considered when adjusting the stringency of a particular hybridization reaction according to methods known in the art.
  • Conditions of low stringency may be 5 as follows (see, also, Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78,
  • Filters containing DNA are pretreated for 6 h at 40 °C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA.
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% 0 BSA, 100 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm
  • 32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55 °C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 °C and re-exposed c to film.
  • Conditions of high stringency may be as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65 °C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%) Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA. Washing of Q filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 min before autoradiography.
  • Nucleic acids used in conjunction with the device of the invention are often oligonucleotides ranging from 10 to about 50 nucleotides in length.
  • an oligonucleotide is 10 nucleotides, 15 nucleotides, 20 nucleotides or 50 nucleotides in length.
  • An oligonucleotide can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, or single-stranded or double-stranded, or partially double-stranded.
  • An o oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, or a combination thereof.
  • An oligonucleotide may include other appending groups, such as biotin, fluorophores, or peptides.
  • An oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
  • D-mannosylqueosine 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, and 2,6-diaminopurine.
  • An oligonucleotide may comprise at least one modified phosphate backbone selected from the group including but not limited to a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • An oligonucleotide or derivative thereof used in conjunction with the methods of this invention may be synthesized using any method known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate ohgonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16, 3209)
  • methylphosphonate ohgonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Nat'l Acad. Sci. U.S.A. 85, 7448-7451), etc.
  • An oligonucleotide may be an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (see Gautier et al., 1987, Nucl. Acids Res. 15, 6625-6641).
  • Ohgonucleotides may be synthesized using any method known in the art
  • reagents for synthesis may be obtained from any one of many commercial suppliers.
  • Spacer phosphoramidite molecules may be used during oligonucleotide synthesis, e.g., to bridge sections of ohgonucleotides where base pairing is undesired or to position labels or tags away from an oligonucleotide portion undergoing base pairing.
  • the spacer length can be varied by consecutive additions of spacer phosphoramidites.
  • Spacer phosphoramidite molecules may be used as 5'- or 3'- oligonucleotide modifiers.
  • Spacer Phosphoramidite 9 i.e., 9-O-Dimethoxytrityl-triethyleneglycol, 1- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Spacer Phosphoramidite 18 i.e., 9-O-Dimethoxytrityl-triethyleneglycol, 1- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Spacer Phosphoramidite 18 i.e., 9-O-Dimethoxytrityl-triethyleneglycol, 1- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Other spacers are available for use in standard oligonucleotide synthesis.
  • Spacer Phosphoramidite C3 and dSpacer Phosphoramidite can be used to destabilize undesirable self-hybridization events within capture ohgonucleotides or to destabilize false hybridization events between incorrectly-matched template/probe complexes.
  • Such spacers when positioned at the 3' end of an oligonucleotide, will also prevent incorrect extension products from being generated when included in a PCR reaction mixture.
  • Spacer Phosphoramidite C3 i.e., 3-O-Dimethoxytrityl-propyl-l -[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Spacer Phosphoramidite C3 3-O-Dimethoxytrityl-propyl-l -[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • a branching spacer may be used as one method to increase label incorporation into an oligonucleotide. Such a branching spacer may also be used to increase a detectable signal by hybridization through multiply branched capture probes or PCR primers. Branching spacers are available commercially, e.g., from Glen Research.
  • Biotinylated ohgonucleotides are well known in the art.
  • An oligonucleotide may be biotinylated using a biotin-NHS ester procedure.
  • biotin may be attached during oligonucleotide synthesis using a biotin phosphoramidite (Cocuzza, 1989, Tetrahed. Lett. 30, 6287-6290).
  • biotin phosphoramidite available from Glen Research is l-Dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-
  • Another 5'-biotin phosphoramidite namely [l-N-(4,4'-Dimethoxytrityl)- biotinyl-6-aminohexyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite, may be used to biotinylate an oligonucleotide.
  • This compound is sold by Glen Research under license from Zeneca PLC.
  • Fluorescent dyes may also be incorporated into an oligonucleotide using dye-labeled phosphoramidites. Two such labels are 5'-Hexachloro-Fluorescein Phosphoramidite (HEX), and 5'-Tetrachloro-Fluorescein Phosphoramidite (TET), both available from Glen Research. 5.7.4 PRODUCTION OF LABELED OLIGONUCLEOTIDES
  • Ohgonucleotides may be labeled with a wide variety of lables for use in the various embodiments of the invention.
  • European Patent Publication No. EP 5 0370 694 A2 entitled, “Diagnostic Kit and Method Using a Solid Phase Capture Means For Detecting Nucleic Acid", by Burdick and Oakes, publication date May 30, 1990, discloses methods of linking labels to ohgonucleotides.
  • a heterobifunctional crosslinking reagent is used to link a synthetic peptide having an N- terminal lysine residue to a 5'-thiol-modified oligonucleotide.
  • Such a crosslinking reagent is N-maleimido-6-aminocaproyl-(2'-nitro, 4'-sulfonic acid) phenyl ester (mal-sac-HNSA).
  • the sodium salt of mal-sac-HNSA is available from Bachem Bioscience.
  • reaction of the mal-sac-HNSA crosslinker with an amino group releases a dianion phenolate (i.e. l-hydroxy-2-nitro-4-benzene sulfonic acid).
  • This dianion phenolate is also a yellow chromophore.
  • the chromophore feature provides (i) a means for quantifying the extent of completion of the coupling reaction (where greater yellow color intensity corresponds to a more complete coupling reaction), and (ii) an aid in monitoring the extent of separation of 5 an activated peptide (i.e. a peptide crosslinked to mal-sac-HNSA and ready for contacting with a 5'-thiol-modified oligonucleotide) from free crosslinking reagent during gel filtration.
  • a mal-sac-HNSA crosslinker may be as follows. First, a peptide is synthesized having an N-terminal lysine. Alternatively, a peptide having an internal lysine may be used since the lysine epsilon amino group is Q actually more reactive than the lysine alpha amino group. Second, an oligonucleotide is synthesized having a 5'-thiol group using methods known in the art. Third, the peptide is reacted with an excess of mal-sac-HNSA in a sodium phosphate buffer (pH 7.1).
  • the peptide-mal-sac conjugate is separated from free crosslinker and the buffer is exchanged to sodium phosphate (pH 6) using a gel filtration column (e.g. NAP-5, Pharmacia, Uppsala, 5 Sweden).
  • a thiol-modified oligonucleotide is activated, desalted and buffer- exchanged to sodium phosphate (pH 6) on a gel filtration column.
  • the activated peptide is reacted with the thiol-modified oligonucleotide.
  • the peptide- oligonucleotide conjugate is purified by ion exchange chromatography (e.g. Nucleogen DEAE-500-10 or equivalent). The elution order from the ion exchange column is as o follows: free peptide first, pep tide-labeled oligonucleotide next, and free oligonucleotide last.
  • Antibodies of use with the methods of this invention include any antibodies 5 known in the art. Such antibodies may be used, for example, to manipulate the nucleic acids of interest.
  • a nucleic acid may be manipluated by antibody binding to the nucleic acid itself or to an antigen (e.g., a protein, peptide or hapten) which is bound (either covalently or non-covalently) to the nucleic acid.
  • nucleic acids are manipulated using peptide antigens covalently attached to PCR primers.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric and humanized antibodies, as described below.
  • polyclonal antibodies which may be used with the invention are 5 heterogeneous populations of antibody molecules derived from the sera of immunized animals.
  • Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest.
  • the production of polyclonal antibodies various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, etc.
  • adjuvants Q may be used to increase the immuno logical response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Monoclonal antibodies which may be used with the invention are homogeneous populations of antibodies to a particular antigen.
  • a monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. o These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), and the more recent human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4, 72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, Inc., pp. 77-96).
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, 5 IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.
  • Monoclonal antibodies which may be used with the methods of the invention include but are not limited to human monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. 0 Nat'l Acad. Sci. U.S.A. 80. 7308-7312; Kozbor et al. 1983. Immunology Today 4. 72-79: Olsson et al., 1982, Meth. Enzvmol. 92, 3-16).
  • a chimeric antibody may be used with the methods of the invention.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a 5 human immunoglobulin constant region.
  • Various techniques are available for the production of such chimeric antibodies (see, e.g., Morrison et al., 1984, Proc. Nat'l Acad. Sci. U.S.A. 81, 6851-6855; Neuberger et al., 1984, Nature, 312, 604-608; Takeda et al., 1985, Nature. 314, 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of Q appropriate biological activity.
  • humanized monoclonal antibody may be used with the methods of the invention.
  • humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarily determining regions
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarily determining regions (CDRs). The extent of the framework region and CDRs have been precisely
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region together via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2
  • Fab expression libraries may be constructed (Huse et al., 1989, Science. 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • the single-lettter amino acid code corresponds to the three-letter amino acid code of the Sequence Listing set forth hereinbelow, as follows: A, Ala; R, Arg; N, Asn; D, Q Asp; B, Asx; C, Cys; Q, Gin; E, Glu; Z, Glx; G, Gly; H, His; I, He; L, Leu; K, Lys; M, Met; F, Phe; P, Pro; S, Ser; T, Thr; W, Trp; Y, Tyr; and V, Val.
  • Suitable antibodies for use with the methods of this invention include the following, available from Affinity Bioreagents, Inc., 79, rue des Morillons, 75015, Paris, France. 5
  • antibodies for use with the methods of this invention may be obtained from Medical & Biological Laboratories Co., Ltd., 440 Arsenal Street, Watertown, Massachusetts 02171, U.S.A.

Abstract

L'invention concerne généralement des procédés de marquage, de tri et de criblage de populations d'acides nucléiques. Elle porte notamment sur un procédé de tri et de comparaison de populations complexes d'acides nucléiques, telle que des banques d'ADNc. Ces populations complexes peuvent être dérivées de cellules ou de types de tissus comportant des variations du phénotype présentant un éventuel intérêt clinique. Le procédé est généralement appelé VG-PLOSM, procédé à Oligonucléotides à marquage peptidique, ou et implique l'utilisation d'étiquettes peptidiques identifiables, liées à des amorces olignonucléotidiques identiques.
PCT/US1999/023906 1998-10-16 1999-10-15 Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique WO2000023622A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
KR1020017004579A KR20010102909A (ko) 1998-10-16 1999-10-15 펩티드-표지된 올리고뉴클레오티드를 이용하여 복합성핵산 개체군을 조작하는 방법
CA002344625A CA2344625A1 (fr) 1998-10-16 1999-10-15 Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique
JP2000577329A JP2002527118A (ja) 1998-10-16 1999-10-15 ペプチド標識オリゴヌクレオチドを用いた複合核酸集団の操作方法
AU11130/00A AU1113000A (en) 1998-10-16 1999-10-15 Methods for manipulating complex nucleic acid populations using peptide-labeled oligonucleotides
EP99954899A EP1121470A1 (fr) 1998-10-16 1999-10-15 Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique

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US17432898A 1998-10-16 1998-10-16
US09/174,328 1998-10-16

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Publication number Priority date Publication date Assignee Title
US6824981B2 (en) * 2000-08-11 2004-11-30 Agilix Corporation Ultra-sensitive detection systems using alterable peptide tags
WO2010127400A1 (fr) * 2009-05-08 2010-11-11 Walter And Eliza Hall Institute Of Medical Research Modulation de la liaison de l'oxyde nitrique synthase inductible (inos) à des peptides socs-box (ssb) contenant des domaines spry
WO2023052629A3 (fr) * 2021-09-30 2023-05-11 Illumina Cambridge Limited Méthodes de blocage

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KR101110013B1 (ko) * 2007-10-05 2012-02-29 (주)바이오니아 서열 내에 어베이직 부분을 포함하는 ｐｃｒ 증폭용프라이머

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WO1997043443A1 (fr) * 1996-05-14 1997-11-20 Boehringer Mannheim Gmbh Procede d'identification et/ou de quantification de l'expression de molecules d'acide nucleique dans un echantillon
US5804382A (en) * 1996-05-10 1998-09-08 Beth Israel Deaconess Medical Center, Inc. Methods for identifying differentially expressed genes and differences between genomic nucleic acid sequences
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WO1989001526A1 (fr) * 1987-08-07 1989-02-23 Genelabs Incorporated Librairie et procede de clonage par coincidence
US5804382A (en) * 1996-05-10 1998-09-08 Beth Israel Deaconess Medical Center, Inc. Methods for identifying differentially expressed genes and differences between genomic nucleic acid sequences
WO1997043443A1 (fr) * 1996-05-14 1997-11-20 Boehringer Mannheim Gmbh Procede d'identification et/ou de quantification de l'expression de molecules d'acide nucleique dans un echantillon
WO1999036575A1 (fr) * 1998-01-15 1999-07-22 Valigene Corporation Vgid multiplex

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6824981B2 (en) * 2000-08-11 2004-11-30 Agilix Corporation Ultra-sensitive detection systems using alterable peptide tags
WO2010127400A1 (fr) * 2009-05-08 2010-11-11 Walter And Eliza Hall Institute Of Medical Research Modulation de la liaison de l'oxyde nitrique synthase inductible (inos) à des peptides socs-box (ssb) contenant des domaines spry
WO2023052629A3 (fr) * 2021-09-30 2023-05-11 Illumina Cambridge Limited Méthodes de blocage

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CN1342208A (zh) 2002-03-27
CA2344625A1 (fr) 2000-04-27

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