[go: up one dir, main page]

WO2025058951A1 - Procédés d'établissement de profils de répertoires immunitaires - Google Patents

Procédés d'établissement de profils de répertoires immunitaires Download PDF

Info

Publication number
WO2025058951A1
WO2025058951A1 PCT/US2024/045631 US2024045631W WO2025058951A1 WO 2025058951 A1 WO2025058951 A1 WO 2025058951A1 US 2024045631 W US2024045631 W US 2024045631W WO 2025058951 A1 WO2025058951 A1 WO 2025058951A1
Authority
WO
WIPO (PCT)
Prior art keywords
probes
cell
target
amplification
cells
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
PCT/US2024/045631
Other languages
English (en)
Inventor
Sanjay Tyagi
Fred R. Kramer
Salvatore A. E. Marras
Ryan J. DIKDAN
Stan KWANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rutgers State University of New Jersey
Original Assignee
Rutgers State University of New Jersey
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 Rutgers State University of New Jersey filed Critical Rutgers State University of New Jersey
Publication of WO2025058951A1 publication Critical patent/WO2025058951A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • This invention relates to methods for profiling immune repertoires and more particularly, to profiling T-cell receptor repertoires and B-cell receptor repertoires. This invention also relates to diagnosis of disease conditions in which the immune cell repertoire is altered.
  • BACKGROUND OF THE INVENTION Two major components of the immune system, B cells expressing B-cell receptors (antibodies) and T cells expressing T-cell receptors (TCRs), recognize highly diverse antigens through the creation of a large repertoire of gene sequences that are not present in the germ line that a person inherits.
  • V variable
  • D diversity
  • J joining
  • this disclosure addresses the above need in a number of aspects.
  • this disclosure provides a method for profiling T-cell or B-cell receptor immune repertoires from multiple T cells or B cells possessing nucleic acid templates that encode chains of T-cell or B-cell receptors.
  • the receptors comprise a T-cell receptor
  • the plurality of primer pairs comprise a first primer pair set that is capable of hybridizing to and amplifying a target region in the ⁇ chain encompassing both the V ⁇ region and the J ⁇ region, a second primer pair set that is capable of hybridizing to and amplifying a target region in the ⁇ chain encompassing both the V ⁇ region and the J ⁇ region; and these primer pair sets are chosen such that essentially all possible combinations of V ⁇ -J ⁇ in the ⁇ chain and essentially all possible combinations of V ⁇ - J ⁇ in the ⁇ chain are amplified.
  • the nucleic acid templates comprise mRNAs
  • the amplification is performed by including a reverse transcriptase in the reactions.
  • the amplification is asymmetric, producing a larger amount of the strand that is the target of the probes.
  • cell lysis is performed by heating prior to amplification.
  • the plurality of reaction vessels are aqueous droplets.
  • the step of fractionating the cells into the plurality of reaction vessels is performed by emulsification or microfluidics.
  • FIG. 1 A small portion of the alignment that shows high levels of homology between the different gene segments is depicted.
  • the shaded nucleotide residues display at least 75% identity.
  • the target regions of six sloppy molecular beacon probes that can be used to discriminate this set of TCR ⁇ gene segments are shown at the bottom of the figure.
  • SEQ ID NOs of the sequences shown in Figure 3 were assigned according to Table 1.
  • Figure 4 shows a hierarchical clustering dendrogram of the TCR ⁇ V variable region sequences shown in Figure 3. The sequences within each shaded region are more closely related to each other than the sequences between these clusters. For example, these clusters could be used to design four SMBs.
  • a second advantageous aspect of the disclosed method is that information about which pair of ⁇ and ⁇ receptor chains are present in the same T cell is inherently provided by the process. This is not the case for next generation sequencing (NGS) strategies, where during cell lysis and library preparation the information about ⁇ and ⁇ pairing is lost, unless elaborate steps, such as single-cell sequencing, are taken to obtain this information.
  • NGS next generation sequencing
  • Partitions may be produced by any suitable manner (e.g., emulsion, microfluidics, microspray, etc.).
  • the partitions are aqueous droplets suspended in an oil matrix, and in other embodiments, the partitions are microwells.
  • the term “droplet” refers to a small volume of liquid that is immiscible with its surroundings (e.g., gases, liquids, surfaces, etc.).
  • a droplet may reside upon a surface that is encapsulated by a fluid with which it is immiscible (e.g., the continuous phase of an emulsion).
  • a droplet is typically spherical or substantially spherical in shape, but may be non-spherical.
  • a droplet may be a “simple droplet” or a “compound droplet,” wherein one droplet encapsulates one or more additional smaller droplets.
  • the volume of a droplet and/or the average volume of a set of droplets provided herein may be less than about one hundred microliters.
  • the diameter of a droplet and/or the average diameter of a set of droplets provided herein is typically less than about one millimeter.
  • Droplets may be formed by any suitable technique (e.g., emulsification, microfluidics, injection, etc.), and those droplets may be monodisperse (e.g., substantially monodisperse) or polydisperse.
  • the cells may be lysed in each partition by heating, such as at about 92 oC.
  • the initial denaturation step renders the target nucleic acids accessible for amplification.
  • the time devoted to this step can be increased to ensure efficient lysis.
  • mild detergents or other lysis reagents, that do not negatively impact amplification can be included in the partitions.
  • the other lysis reagents include proteinase K. The lysis may or may not be proformed at the same time as amplification.
  • the step of amplification is performed by a PCR, recombinase polymerase amplification, transcription-mediated amplification, or strand-displacement amplification.
  • Asymmetric amplification is preferred because it provides better opportunity for probes to bind.
  • amplification is performed by creating cDNA copies from the mRNAs, and in other embodiments, the DNA is directly amplified.
  • asymmetric PCR refers to the preferential PCR amplification of one strand of a DNA target by adjusting the molar concentration of the primers in a primer pair so that they are unequal.
  • An asymmetric PCR assay produces a predominantly single-stranded product and a smaller quantity of a double-stranded product as a result of the unequal primer concentrations.
  • the lower concentration primer is quantitatively incorporated into a double-stranded DNA amplicon, but the higher concentration primer continues to prime DNA synthesis, resulting in continued accumulation of a single-stranded product.
  • Amplification may also include isothermal amplification.
  • isothermal means conducting a reaction at a substantially constant temperature, i.e., without varying the reaction temperature in which a nucleic acid polymerization reaction occurs.
  • TCRs consisting of the combination of the former are called ⁇ TCRs, and TCRs consisting of the combination of the latter are called ⁇ TCRs.
  • T cells having such TCRs are respectively called ⁇ T cells and ⁇ T cells.
  • the TCRs are structurally very similar to a frequent antibody binding region (Fab fragment) of an antibody produced by B cells, and they recognize antigen molecules in concert with a major histocompatibility (MHC) molecule. Since the TCR gene of each mature T cell has undergone gene rearrangement, an individual person has highly diverse TCRs that enable recognition of a very large number of different antigens
  • Each TCR chain is comprised of a variable domain (V) and a constant domain (C).
  • a constant domain has a short cytoplasm section penetrating the cell membrane.
  • a variable domain is present outside the cell and binds to an antigen-MHC complex.
  • a variable domain has three hypervariable domains or regions called complementarity- determining regions (CDRs), which bind to an antigen-MHC complex.
  • the three CDRs are called CDR1, CDR2, and CDR3.
  • the gene rearrangements in TCRs are similar to the gene rearrangements in B-cell receptors, which are known as immunoglobulins.
  • the organization of the B-cell receptor (BCR) on B cells (antibodies) is similar to the organization of the T-cell receptor on T cells.
  • each of the plurality of primer pairs hybridize to and amplify different target regions in the nucleic acid sequence of a cell receptor (e.g., TCR or BCR).
  • the nucleic acid molecule encoding the cell receptor comprises a cell receptor gene or an mRNA.
  • the nucleic acid molecule encoding the cell receptor is a cDNA of the mRNA encoding the cell receptor.
  • a “target region,” “target nucleic acid sequence,” or “target sequence” refers to a specific sequence that may include all or part of a longer sequence that needs to be identified.
  • the target region comprises one or more regions in a V segment and/or a J segment of the cell receptor (e.g., TCR or BCR).
  • the target region comprises one or more regions in the ⁇ chain and/or the ⁇ chain of a T-cell receptor.
  • one or more regions are located in the V ⁇ segment, the V ⁇ segment, the J ⁇ segment, and/or the J ⁇ segment of the T-cell receptor.
  • one or more regions are located in the complementarity determining region 3 (CDR3) of the T-cell receptor.
  • CDR3 complementarity determining region 3
  • a set of probes are employed that consist of multiple subsets of probes, where different subsets fluoresce in different fluorescent colors. Each subset may contain from 1 to 10 different probes. In some embodiments, fewer or more probe subsets may be employed.
  • the cell receptor is a TCR, and its ⁇ chain is amplified with one set of primers and its ⁇ chain is amplified with a second set of primers generating an ⁇ amplicon and a ⁇ amplicon, respectively.
  • each of these amplicons contains the V and J regions present in the respective ⁇ or ⁇ genes present in the cell which is being analyzed.
  • the V region of the ⁇ amplicon is detected using one subset of probes labeled in one color
  • the J region of the ⁇ amplicon is detected using a second subset of probes labeled with a second color.
  • the V region of the ⁇ amplicon is detected using a third subset of probes labeled in a third color
  • the J region of the ⁇ amplicon is detected using a fourth subset of probes labeled with a fourth color.
  • Probes refers to an oligonucleotide capable of binding to a target nucleic acid having a complementary sequence. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. There may be any number of base pair mismatches, which will interfere with hybridization between the target sequence and the single-stranded nucleic acids described herein. However, if the number of mismatches is so great that no hybridization can occur under Docket No.: RU 2023-126/FR: 096747.00494 even the least stringent of hybridization conditions, the probe is not a complementary target sequence.
  • the probes according to this disclosure are self-reporting probes.
  • probes can be made in various detection formats, such as dual labeled probes, including linear probes, TaqMan probes, molecular beacon probes, and sloppy molecular beacon (SMB) probes.
  • a “sloppy” probe refers to a probe that is mismatch- tolerant. Mismatch-tolerant probes may hybridize with more than one target sequence to generate a detectable signal at a detection temperature in an assay, and various hybrids so formed will have different melting temperatures. Examples of such probes are hairpin or linear probes with a fluorescent moiety whose level of fluorescence increases upon. See, e.g., U.S. Pat. Nos.5,925,517 and 7,662,550.
  • the sloppy probes are dual-labeled hairpin probes or molecular beacon probes, described in U.S. Pat. Nos.5,925,517 and 7,662,550.
  • These hairpin probes contain a target binding sequence flanked by a pair of arms complementary to one another. They can be DNA, RNA, or PNA, or a combination of all three types of nucleotides. Furthermore, they can contain modified nucleotides and modified internucleotide linkages. They can have a first fluorophore on one arm and a second fluorophore on the other arm, wherein the absorption spectrum of the second fluorophore substantially overlaps the emission spectrum of the first fluorophore.
  • a portion or the entirety of the arm sequences of the probe can have complementarity to the target sequence, and a portion of the target region of the hairpin probe can participate in forming the hairpin along with one or both arm sequences.
  • Probes according to this disclosure need not possess complementary arms. Docket No.: RU 2023-126/FR: 096747.00494
  • sloppy molecular beacons can be used in assays to detect the presence of one variant of a nucleic acid sequence segment of interest from among a number of possible variants or even to detect the presence of two or more variants. These probes can therefore be used in combinations of two or more in the same assay.
  • a first probe may bind strongly to a wild-type sequence, moderately to a first allele, weakly to a second allele and not at all to a third allele; while a second probe may bind weakly to the wild-type sequence and the first variant, and moderately to the second variant and the third variant.
  • Additional sloppy molecular beacon probes will exhibit yet different binding patterns due to their different target binding sequences.
  • the patterns of the fluorescence emission spectra from combinations of sloppy molecular beacon probes can define different related, but different, sequences.
  • Sloppy molecular beacon probes have the ability to bind to a number of different related target sequences at a low temperature, where they fluoresce brightly. However, as the temperature is raised, the different hybrids formed by the sloppy molecular beacon dissociate at different temperatures, depending on the degree of complementarity, resulting in a reduction of fluorescence intensity. The fewer the number of mismatches in the hybrid, the higher the melting temperature is.
  • the melting profiles Docket No.: RU 2023-126/FR: 096747.00494 obtained in each fluorescent color will independently identify different targets in each of a number of different groups, where each different group is targeted by a differently colored the probe set.
  • These multiplex assays have the ability to distinguish a vast number of distinctive targets in the same assay. For instance, an assay simultaneously utilizing four differently colored subsets of sloppy molecular beacon probes can distinguish as many as 3,000,000 different immune signatures that can be present in T cells.
  • the plurality of probe-amplicon hybrids have a melting temperature of about 80°C for probes that do not form mismatches with the target region and at about 25 oC for probes that form the maximum number of mismatches with the target region.
  • the probes may have an internal stem, and in other embodiments, the probes may have a terminal stem. The stem length can range from 2 to 10 base pairs.
  • adjacent probes that bind to the same target near each other and then interact via fluorescence resonance energy transfer (FRET) or contact-mediated quenching can also be used in the disclosed method. Examples of such probes may include Lights-On/Lights- Off probes described in M. G.
  • the set of probes comprises a probe having a chemical modification, such as a modified nucleotide, a modified chemical backbone, or a combination thereof.
  • a “chemical modification” refers to a chemical difference in a compound when compared to a naturally occurring counterpart.
  • Chemical modifications of oligonucleotides may include nucleoside modifications (such as sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.
  • an “internucleoside linkage” refers to a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • a chemical modification does not include differences only in the nucleobase sequence. Examples of modifications may include, without limitation, a 2’-O-methyl-modified sugar moiety.
  • Probes may consist of ribonucleotides instead of deoxyribonucleotides. Probes may also contain both ribonucleotides and deoxyribonucleotides. In some embodiments, the probes have a fluorophore and a quencher. The probes may have other labels as described in U.S. Pat. No.5,925,517.
  • a “fluorophore” includes a molecule that is capable of absorbing energy at a wavelength range and releasing energy at a wavelength range other than the absorbance range.
  • the fluorophore is a Docket No.: RU 2023-126/FR: 096747.00494 molecule that is capable of absorbing energy at about 250 nm to about 900 nm, and can release energy at a wavelength range of about 260 nm to about 910 nm.
  • excitation wavelength refers to the range of wavelengths at which a fluorophore absorbs energy.
  • emission wavelength refers to the range of wavelengths that the fluorophore releases energy or fluoresces.
  • amplification primers are a pair of nucleic acid molecules that can anneal to 5’ or 3’ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between.
  • a further criterion for the design of the primers is that all possible combinations of V and J should be amplified without bias. Persons skilled in the art will appreciate that the same primers will not amplify non-recombined cell receptor genes, because the distance between the two primer binding sites will be so large that efficient amplification does not occur. These criteria may be satisfied by one pair of primers for each of the a and b chains, or the criteria may be satisfied by using multiple forward or reverse primers for the a and b chains.
  • An example of a suitable primer set has been described by Robins et al.2009 Blood 114, 4099-4107. Other suitable primer sets are discussed by Ch’ng et al.2019 European Journal of Immunology 49, 1186-1199.
  • the immune signatures of single cells are used to obtain immune repertoires of collections of cells.
  • Immune signatures are sets of multiple melting profiles that, in the case of TCR, are obtained from single cells for the V ⁇ , J ⁇ , V ⁇ , and J ⁇ regions, that enable the gene segments that are used in a single cell to be identified.
  • diseases or disorders may include, without limitation, a malignant disease (e.g., cancer), a viral disease (e.g., HIV-1 infection), a bacterial disease (e.g., tuberculosis), a fungal disease, a protozoan disease, a parasitic disease, an allergic disease, or an autoimmune disease.
  • a malignant disease e.g., cancer
  • a viral disease e.g., HIV-1 infection
  • bacterial disease e.g., tuberculosis
  • a fungal disease e.g., tuberculosis
  • a protozoan disease e.g., tuberculosis
  • one or more characteristics of the immune repertoire of a subject is compared to the immune repertoire of reference samples obtained from other subjects having a certain disease condition.
  • the subject can be a human or an animal. Docket No.: RU 2023-126/FR: 096747.00494
  • a “sample” refers to any biological fluid or tissue obtained from a patient that contains cells whose receptor arrangement needs to be determined.
  • the sample may be a “clinical sample,” which is a sample derived from a subject, such as a human patient or veterinary subject.
  • the samples may also include sections of tissues, such as frozen sections taken for histological purposes.
  • compositions and kits for obtaining immune signatures from cells, for determining immune repertoires from a collection of cells, and for performing clinical diagnostic assays.
  • These compositions and kits include instructions and reagents needed to perform the assays.
  • the reagents include primer sets, probe sets, and amplification reagents, including polymerases, nucleotides, and buffers.
  • the reagents may also include oils and detergents for droplet generation, reagents for cell lysis. These reagents can be provided separately or in various combined mixtures. Additional Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
  • the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
  • the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.
  • the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.
  • the terms “and/or” or “/” means any one of the items, any combination of the items, or all of the items with which is associated.
  • the word “essentially” or “substantially” does not exclude “completely,” e.g., a composition that is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of this disclosure.
  • the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.
  • the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure.
  • Example 1 An Exemplary Process for Obtaining T-cell Repertoires from T cells Aligning and Choosing Target Sequences
  • the first step was to download all V, D, and J sequences present in the human T-cell receptor ⁇ and ⁇ chains from a gene sequence repository.
  • An example of such a repository is Ensembl (Cunningham et al.2022 Nucleic Acids Research 50, D988-995). The sequences within each group were then aligned using a multi-sequence alignment tool.
  • SMBs Sloppy molecular beacons
  • these probes When these probes bind to a fully or partially complementary target, their fluorescence is increased. They contain a fluorophore and a quencher moiety, for example, at their terminals. However, it is possible to attach these labels at internal locations and obtain good background-to-signal ratios, i.e., the fluorescence intensity displayed upon binding to the target divided by the fluorescence intensity before binding.
  • Their probe lengths may vary from 10 to 100 nucleotides, preferably 10 to 40 nucleotides, and their stem lengths may vary from 2 to 10 nucleotides. Under certain situations, it may be possible to design probes that do not have any stem. Such situations may include when the target itself has internal stems, which results in the probe sequence also having internal stems.
  • Such probes do not possess a designed stem, but rather an internal stem serves to bring the fluorophore and the quencher close to each other in the unbound form of the probe.
  • Another situation where a designed stem may not be necessary is when the fluorophore and the quencher have a high affinity for each other, and they tend to bind to each other when present in the same molecule.
  • sloppy molecular beacons may or may not possess a terminal stem. Under certain conditions, it may be advantageous to use adjacent probes that bind to the same target near each other and then interact via fluorescence resonance energy transfer (FRET) or contact-mediated quenching.
  • FRET fluorescence resonance energy transfer
  • SMBs can consist of natural nucleotides with a natural phosphodiester backbone, or they can contain modified nucleotides and/or modified chemical backbones.
  • the modified nucleotides may be designed to increase or decrease the specificity of complementary base recognition.
  • Docket No.: RU 2023-126/FR: 096747.00494 Designing Sloppy Molecular Beacon Probes for T-cell Receptor Regions To discriminate all of the targets in a given region of variability, such as the V region of TCR ⁇ discussed above, a set of 2 to 8 SMBs were designed using computer programs.
  • each SMB should bind to a large fraction of the targets in the target set, and that the strengths of their binding should vary from target to target and not be the same. The presence of more mismatches results in weaker probe-target hybrids, and the presence of a lesser number of mismatches results in stronger probe-target hybrids.
  • a second consideration in the choice of the sequences present in the set of SMBs is that those sequences in the target region that do not bind well to one SMB in the set should bind well to one or more other SMBs in the set. In fact, the set of all SMBs that are chosen should be such that all targets are covered by one or more SMB.
  • an SMB can have a target that is fully complementary to it, that is not a necessity. Indeed, SMBs can be chosen that are not fully complementary to any target sequence within the target region. These objectives are achieved by choosing appropriate lengths of the target binding region of the SMBs, and then by choosing appropriate locations within the targets to which the SMBs bind. Although the length of the SMB’s target-binding region can vary from 10 to 50 nucleotides, a region was selected that yields probe-target hybrids melting at about 80 oC for probes that do not form mismatches, and at about 25 oC with targets whose probes form the maximum number of mismatches in the group of target sequences.
  • the prediction of the melting temperature of each SMB with each target is determined with the aid of computer programs, such as Primer3 (Schgasser et al. 2012 Nucleic Acids Research 40, e115) and the DINAMelt web server (Markham and M. Zuker 2005 Nucleic Acids Research 33, W577-581).
  • Primer3 Greek Gasser et al. 2012 Nucleic Acids Research 40, e115
  • DINAMelt web server Markham and M. Zuker 2005 Nucleic Acids Research 33, W577-581
  • Short stems melt at low temperatures and present little impediment for binding of the SMB to the target, while longer stems melt at higher temperatures and present a stronger impediment for binding to the target.
  • Docket No.: RU 2023-126/FR: 096747.00494 The manner in which stem length and stem strength impact the function of SMBs, and their coverage of a full set of target sequences, is discussed in Example 3.
  • longer stems create impediments to binding to targets, especially for SMBs forming many mismatches with a target sequence, yet small stems enable binding to such targets.
  • the computer program examines all possible combinations of SMBs (for example, a set of 6 SMBs) and scores the set for producing maximally dispersed melting temperature distributions for the entire group of target sequences. The set of SMBs that produces the best score for this parameter is then selected for use.
  • the group of target sequences (such as those shown for V ⁇ regions in Figure 3) were first hierarchically clustered using a clustering program such as MEGA (Tamura et al. 2021 Molecular Biology and Evolution 38, 3022-3027).
  • Hierarchical clustering analysis produces dendrograms that group similar sequences together and reveal the “evolutionary” relationships between the sequences. An example of such a dendrogram is shown in Figure 4.
  • Oligonucleotides corresponding to the portion of the gene that is the target of the designed SMBs were obtained from a custom oligonucleotide manufacturer. Each target oligonucleotide was then separately mixed with each SMB under salt and buffer conditions that were similar to the buffer conditions found in the PCR assay that was planned to be used. A slight molar excess of the target (2- to 10- fold) was utilized in these assays.
  • the melting profiles (also referred to as thermal denaturation profiles) of the hybrids by the binding of each SMB to each target were then determined in a real-time PCR instrument that monitors the fluorescence of hybridization reactions while raising the temperature.
  • each of the 48 TCR ⁇ sequences listed in Figure 3 was mixed with each of the four SMBs (SMB 1, 2, 3, and 4; all having 2 nucleotide-long stems) whose sequences are shown in Table 1.
  • SMB 1, 2, 3, and 4 all having 2 nucleotide-long stems
  • a comparison of the TRBV13 curves across panels demonstrates how the same target, when probed by different SMBs, yields very different melting profiles.
  • One of the key aspects of the disclosed method is to use the combined set of SMBs in order to obtain a multiplex profile that characteristically identifies the target. Since multiple SMBs interrogate different aspects of the target sequence, their combined information is more accurate than that of each SMB by itself. Docket No.: RU 2023-126/FR: 096747.00494 To illustrate the great diversity of melting temperatures that these sets of targets can yield with four different SMBs, the melting temperatures are shown for all targets and all molecular beacons on the same plot ( Figure 6).
  • the melting temperatures yielded by the four molecular beacons for TRBV6-7 are different from those yielded by TRBV6-1 to TRBV6-6.
  • minute changes in the sequences result in detectable changes in the set of melting profiles.
  • the probe sequence of one of the molecular beacons can be changed so that it targets another region of that TCR sequence where the target sequences diverge from each other, or an additional SMB can be added. This can be accomplished by shifting the target region to the right by 9 nucleotides.
  • SMB 5 whose sequence is shown near the bottom in Figure 3.
  • SMB 5 can either be used as a replacement for one of the four SMBs in the set, or it can be added to the set. Ensuring a dependable approach in designing SMBs for specific targets is crucial. Therefore, a correlation between the melting temperatures of the SMBs predicted by the DINAMelt computer program to those observed experimentally was assessed. The details are presented in Example 5.
  • Figure 7 compares the melting temperatures determined experimentally with the melting temperatures predicted by the DINAMelt web server. The results demonstrated that there is a significant correlation between the two sets of melting temperatures, indicating that SMB sets can be designed bioinformatically without the need for extensive trials and testing.
  • Another aspect of this invention is to use multiple SMBs for the same set of targets (exemplary sets are TCR ⁇ , ⁇ , V, or J) together in a multiplex reaction to create high-content Docket No.: RU 2023-126/FR: 096747.00494 melting profiles.
  • the set of SMBs yielded a compound melting profile for a given target in which the profiles of individual SMBs are superimposed on top of each other within the same curve. If the melting transitions of different SMBs for the same target are sufficiently separated from each other within the temperature range, they can clearly be resolved when seen in the negative first derivative plot.
  • the melting transitions are close to each other, they may not be resolved. However, in both cases, a uniquely distinguishable profile is created for each target.
  • An example of such compounding is illustrated in Figure 8. Gray curves at the top of Figure 8 represent four melting profiles for TRBV13 with SMBs 1, 2, 3, and 4 individually, and the black curve is the melting profile when the four SMBs were combined in one reaction. The negative first derivative plots of these profiles are presented at the bottom of Figure 8, which shows that in the compound profile, two of the four component melting transitions are clearly identifiable (from SMB 2 and SMB 4), whereas the melting transitions from the other two SMBs (1 and 3) appear to be compressed together.
  • the compound profile is information-rich, as it encompasses the response of each of the four probes that have their own separately distinctive discriminatory capacities.
  • the details of how high-content compound melting profiles were created are described in Example 6.
  • the diversity of the high-content compound profiles and their negative first derivative curves created by multiple SMBs used in a multiplex for all 48 targets and controls is shown in Figure 9.
  • a visual examination suggests that these profiles are distinct from each other.
  • a computer analysis needed to be performed. Such an analysis is presented below.
  • Another key aspect of this invention is a system and method for developing machine learning models to identify target sequences from the melting profiles that are generated by the hybridization of sets of SMBs.
  • the method involves obtaining multiple replicates of melting profiles from a set of multiple SMBs for one or more gene segments, as described above. Examples of such profiles are presented in Figure 9. These replicates were obtained by carrying out repeated experiments that obtained the melting profiles from the same set of targets and SMBs. These replicates introduced probabilistic variabilities that are expected in actual assays.
  • the raw temperature vs. fluorescence curves recorded by the instrument were then processed to obtain the negative first derivatives, which are used for training machine learning algorithms. Alternatively, smoothed fluorescence vs. temperature curves can be utilized directly.
  • Example 7 provides details of the data processing needed to implement machine learning- based identification of targets from profiles and discrimination between profiles of different targets. Briefly, the data from the negative first derivative curves, or from smoothed primary curves, was compiled into a spreadsheet, where each row represents the negative first derivative curves for a given target determined over the full range of temperatures. The last element in each row indicated the name of the target whose data appears in that row. The spreadsheet contained multiple rows for each target corresponding to the replicates. Multiple machine learning algorithms, such as the “Subspace Discriminator” in the “Ensemble Classifier” group, were trained by utilizing the data prepared in MATLAB. The predictor variables were defined as columns containing the melt data for each sample, and the response categories were defined as the target names.
  • the trained model with the highest accuracy was then selected for further analysis.
  • the accuracy of the selected model was demonstrated through a “confusion matrix” plot which visualizes the correct identification of the TCR ⁇ sequences.
  • a confusion matrix plot for the data in Figure 9 is presented in Figure 10 using the Subspace Discriminator algorithm, which resulted in a discrimination accuracy of 96%.
  • the dark gray quadrants in the plot represent correct identifications, while the light gray quadrants indicate misidentification events.
  • the majority of Docket No.: RU 2023-126/FR: 096747.00494 TCR ⁇ sequences were correctly identified along the diagonal, indicating high prediction accuracy.
  • the numbers within the quadrants represent the frequency of correct calls, mostly being 6 (corresponding to the number of replicates used).
  • T cells that are distributed into these partitions can be enriched from the donated blood or from other tissues via affinity purification.
  • affinity purification procedure antibodies against receptors on the T-cell surface, for example, CD3 and CD4, are tethered to magnetic beads and are utilized to selectively capture those cells.
  • the affinity-purified cells are then purified further by flow cytometry before their distribution into partitions.
  • the partitions can be wells in a multi-well plate, aqueous droplets suspended in oil, or components of another suitable partitioning system known in the art.
  • the partitions can be created by emulsification, microfluidics, or by other means known in the art. They may remain suspended in a three-dimensional space, spread on a two-dimensional surface, or passed through channels with a temperature gradient during the steps of amplification and/or detection ( Figure 1).
  • Each partition can further include cell lysis reagents, amplification reagents, detection reagents, and/or sloppy molecular beacon probes along with the cells from the beginning of the procedure.
  • the next step after placement of the single T cells into partitions is the amplification of the recombined TCR genes or the amplification of the mRNAs encoded by the recombined TCR genes. This is accomplished by either PCR, recombinase polymerase amplification, transcription- mediated amplification, strand-displacement amplification, or another amplification technique known to persons skilled in the field.
  • TCR mRNA amplification is advantageous, because it is among the most highly expressed mRNA species in T cells, and these mRNAs do not possess introns. Amplification of these mRNAs is accomplished by including a high-temperature reverse transcriptase in the reaction, which creates a cDNA copy of each mRNA, which then serves as substrates for PCR.
  • Target amplification especially by PCR, usually includes an initial denaturation step, such as at 92 oC, which naturally causes lysis of the cells, and renders the target nucleic acids accessible for amplification. The time devoted to this step can be increased to ensure efficient lysis.
  • Primers that are suitable for amplification of the recombined TCR target genes or target mRNAs are included in the amplification mixture. These primer sets are designed such that portions of the recombined TCR genes, or mRNAs, are amplified for all T cells in their respective partitions, irrespective of which rearranged TCR sequences are present. Furthermore, these primers are designed such that all TCR arrangements are likely to be amplified equally well without bias. Persons skilled in the art will appreciate that the same primers will not amplify non- recombined TCR genes, because the distance between the two primer binding sites is so large that efficient amplification does not occur.
  • CDR3 complementarity determining region 3
  • This region is important for the antigen specificity of the TCR, and therefore holds high functional significance.
  • one of the primers is placed in the constant region that is close to the CDR3 region, and the second primer is placed in the variable region.
  • the primer for the constant region is able to initiate DNA polymerization from all recombined arrangements, whereas the primer for the variable region initiated polymerization only Docket No.: RU 2023-126/FR: 096747.00494 on particular TCR ⁇ V segments.
  • V segment primers are needed at the same time to enable the amplification of all possible rearranged TCR genes.
  • a suitable set of such primers has been described by Robins et al.2009 Blood 114, 4099-4107. Other suitable primer sets are discussed by Ch’ng et al. 2019 European Journal of Immunology 49, 1186-1199.
  • the recombined genes of both the ⁇ and ⁇ chains are amplified simultaneously using a primer set specific for each. When multiple primers are used together, undesirable false amplicons are often produced, due to interactions between various primers. These false amplicons compete with the desired amplicons, and can therefore limit the sensitivity of the assays.
  • RNA residue is placed at a position several nucleotides inward from the 3’ end of the primer and the 3’ end is blocked.
  • primer optimization steps can be used for the amplification step of this invention.
  • multiple V region primers can be replaced with fewer primers, or just one primer, designed to bind to the upstream constant region.
  • this alternative is less desirable, because it will yield rather long amplicons that may be slower to amplify.
  • this invention employs asymmetric amplification, such that the DNA amplicon strands that are complementary to the SMBs are produced in larger amounts than the corresponding complementary strands. This helps to ensure that the SMB targets are abundant and accessible for SMB binding, with little competition from the less abundant complementary amplicons.
  • the disclosed method involves the determination of a melting profile in each partition after amplification.
  • partitions There are at least three different configurations of partitions that can be used to obtain melting profiles from the partitions. These configurations are referred to here as either “one-dimensional flow,” “two-dimensional stationary,” or “three-dimensional stationary.” The first two of these are illustrated in Figure 1.
  • the partitions In the one-dimensional flow mode, which is suitable for aqueous droplets in oil suspension, the partitions are passed over a gradient of temperature as their fluorescence is monitored in multiple channels (Figure 1, right). Each droplet is tracked as it passes over zones of increasing temperature. This can be accomplished, for example, by the use of a linear array of light sources and detectors.
  • the two-dimensional stationary mode is suitable for fixed partitions, such as wells and aqueous droplets suspended in oil spread over a surface. Here, the entire surface is heated while fluorescence was measured ( Figure 1, left).
  • This system is amenable to image-based recording of fluorescence changes as a function of temperature.
  • the partitions (wells or droplets) can be computationally segmented (i.e., their boundaries are defined), and fluorescence intensity can be determined for each partition as a function of temperature. Examples of such systems are described by Athamanolap et al. (Athamanolap et al. 2019 Analytical Chemistry 91, 12784-12792 and by Luo et al.2022 Small Methods 6, e2200185).
  • the reaction mixtures contain Docket No.: RU 2023-126/FR: 096747.00494 SMB sets for each of these sets of targets.
  • Each SMB set consists of multiple SMBs labeled with the same colored fluorophore, as discussed above, but the SMB sets for different targets have different and distinguishably colored fluorophores.
  • four melting profiles are obtained, one each for TCR V ⁇ , TCR J ⁇ , TCR V ⁇ , and TCR J ⁇ , respectively, for each partition.
  • the specificity for the V and J segments can come from primers designed to be specific for individual V and J segments, or from SMBs that bind to individual V and J segments, or from combinations of the two.
  • primers designed to be specific for individual V and J segments or from SMBs that bind to individual V and J segments, or from combinations of the two.
  • the specificity generating 3’ ends of the V segment-specific primers are placed towards the 5’ end of the variable region ( Figure 11 top) so that sufficient target variability remains in the amplified product. This ensures that the SMBs are able to bind to the variable region, and identify it through their melting profiles.
  • the 3’ end of the V segment-specific primers are placed inwards within the variable region ( Figure 11middle).
  • the 5’ tails of the primers contain tag sequences that “encode” the targets, thereby assigning unique codes to each target.
  • the complements of the codes are arbitrary target sequences that can later be “decoded” by the binding and melting of the SMBs. After amplification, these tags are incorporated into the amplified product and then detected using a set of SMBs that bind to the complement of the encoding sequences ( Figure 11 bottom).
  • Example 7 demonstrates how it is possible to identify essentially all V ⁇ segments using a set of SMBs labeled in one color. The rest of the segments J ⁇ , V ⁇ , and J ⁇ can similarly be identified using SMB sets that are specific to those segments and are labeled with fluorophores of three other distinct fluorescent colors. This results in four independent differently colored melting profiles that taken together report the identity of all four gene segments, V ⁇ , J ⁇ , V ⁇ , and J ⁇ .
  • each profile explicitly identifies a particular target gene fragment, the four profiles together indicate which TCR arrangement is present in a cell. Since specific V ⁇ , J ⁇ , V ⁇ , and J ⁇ arrangements can be identified from 2.9 x 10 6 to 3.3 x 10 6 possible arrangements, the depth of this analysis is very high and covers all-natural diversity except the variability that arises from random nucleotide excisions and additions at the V, D, and J junctions during the recombination process. Since the diversity of T-cell clones is extremely high in blood, the vast majority of T-cell clones are found only once in a sample. Some clones may be expanded, and their expansion may vary from clone to clone.
  • a higher Shannon entropy value indicates a more diverse TCR repertoire, with a larger number of unique TCR sequences, and a relatively even distribution of frequencies. Conversely, a lower Shannon entropy value indicates a less diverse repertoire, with fewer unique TCR sequences and/or an uneven distribution of frequencies.
  • Shannon’s entropy can be used to compare the TCR repertoire diversity between different samples or populations, track changes in this diversity over time, and/or assess the impact of diseases or treatments on a patient’s TCR repertoire. Docket No.: RU 2023-126/FR: 096747.00494 Shannon’s entropy is just one of many measures used in TCR profiling to characterize the diversity and complexity of TCR repertoires.
  • T cells from multiple patients with a similar disease condition such as having a confirmed diagnosis of an autoimmune disease, such as lupus erythematosus, or rheumatoid arthritis, are compared, and the clones that are common to each diagnosis are identified. Since only a few clones are closely associated with the disease state, they can be used as biomarkers for that disease. The disease condition can therefore be diagnosed in assays that focus on these marker clones.
  • a simple way of creating such focused assays for the public clones is to use a subset of primers that are specific to the V ⁇ , J ⁇ , V ⁇ , and J ⁇ segments that are found in these clones in combination with a full complement of SMBs. This will result in amplification of only the public clones, and will not amplify the rest of the repertoire. Therefore, only those partitions that harbor one or more particular public clones will yield positive melting profiles.
  • These focused assays can also be performed on public clones that have been identified previously by other methods such as next-generation sequence analysis. A key advantage of these focused assays is that since the rest of the repertoire is not amplified, the probability of finding and identifying the public clones is significantly increased. Example 2.
  • SMB Sloppy Molecular Beacon
  • probes were resuspended in a 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0 solution at a concentration of 100 ⁇ M and stored at -20 oC until further use.
  • Target oligonucleotides (see Table 1, SEQ ID Nos: 14 to 61) were purchased from Integrated DNA Technologies (Coralville, IA), and were dissolved in RNase-free water at a concentration of 100 ⁇ M. The target oligonucleotide solutions were first diluted in water to a concentration of 10 ⁇ M and then further diluted in water to a working concentration of 1 ⁇ M. The stock and working target oligonucleotide solutions were stored at -20 oC until they were used. Table 1.
  • probe-target hybrid strength is the only determinant of the melting temperature of the hybrid formed by an SMB with its target oligonucleotide.
  • this high “dropout rate” was rectified by decreasing the length of the stem in those molecular beacons.
  • Hybrid melting reactions were carried out using 0.2 ml white polypropylene PCR tubes (USA Scientific, Ocala, FL) in a Bio-Rad CFX96 Real-Time System spectrofluorometric thermal cycler. The thermal cycler was programmed to heat the reactions for 3 min at 95 oC, after which the reactions were allowed to cool passively to room temperature.
  • the thermal cycler was then programed to cool the reactions for 2 min at 4 oC, and Docket No.: RU 2023-126/FR: 096747.00494 to then increase the temperature in steps of 0.5 oC to 95 oC, holding each step for 10 sec while recording the fluorescence intensity.
  • Fluorescence intensity as a function of temperature for each reaction is shown in Figure 12, left. Four plots are shown where the particular SMB that was present in those reaction is indicated. Each curve in these plots stems from one reaction and shows the response to one particular target. For each SMB, the plots are normalized such that at 85 oC the fluorescence intensity is the same.
  • the software of the thermal cycler automatically determines the negative derivative of fluorescence intensity with respect to temperature (-dI/dT) as a function of temperature. These instrument-generated plots are shown in Figure 12, right. From this data, the software automatically determines the melting temperature of the hybrid formed by binding of the SMB to the target oligonucleotide. The melting temperature data for all SMBs and all targets are presented in Figure 13. As described above, the fluorescence intensity in these melting profiles is generally high at low temperatures, because the molecular beacons are bound to their target oligonucleotides, which physically separates their fluorophore from their quencher, resulting in a strong fluorescent signal. As the temperature is raised further, the fluorescence intensity remains high, because the hybrids are stable.
  • probe-target hybrids are “dropouts,” as the SMBs did not bind to these target oligonucleotides (or if they did bind to their target oligonucleotides, the flexibility of the hybrid that they formed containing many mismatches, combined with the greater length of their stem sequences, enabled their fluorophores to interact with their quenchers, significantly decreasing their fluorescence intensity).
  • the number of dropouts was 21, 9, 4, and 3, for stem lengths of 6, 4, 2, and 0 respectively (Figure 13). This decrease indicates that smaller stem lengths in the SMBs yielded melting profiles from a larger number of targets. Overall, these results show that dropout rates decrease significantly when the length of the sloppy molecular beacon’s stem is decreased.
  • Gray curves in Figure 8 top represent four melting profiles for TRBV13 with SMBs 1-4 individually, and the black curve is the melting profile when the four SMBs were combined into one reaction.
  • the negative first derivative plots of these profiles are presented in Figure 8 bottom, which shows that in the compound profile two of the four component melting transitions are clearly identifiable, whereas the melting transitions from the other two SMBs appear to be compressed together. Nonetheless, the compound profile is information-rich, as it encompasses the response of all four sloppy molecular beacon probes, each of which has a different discriminatory capacity.
  • each row indicates the name of the target.
  • this spreadsheet there were 49 rows (48 for each target and one for the no-target control) for each replicate. Therefore, there were Docket No.: RU 2023-126/FR: 096747.00494 49 x 6 rows for an experiment with six replicates. More replicates could also have been used for better predictability.
  • Each row in this spreadsheet consisted of 184 columns, 183 for the values of the negative first derivatives, and the last one for the name of the target.
  • Appropriate machine learning algorithms have been implemented in many computer languages. As an example, the MATLAB programming language was utilized. The Python computer environment can also provide equivalent results. Training data, prepared as described above, was imported into the MATLAB workspace.
  • the targets were chosen based on their expected melting temperatures with SMB1, roughly evenly distributed between 30°C and 65°C ( Figure 5).
  • QX200 droplet generator with DG8 cartridges and droplet generation oil (Bio-Rad)
  • each solution was partitioned into nanoliter droplets.
  • the aqueous mixture of probes and targets passes through a microfluidic channel and merges with an oil stream, producing uniform and stable droplets ( ⁇ 0.9 nL) in an oil matrix.
  • After creating the seven droplet suspensions they were combined into a single mixture. Despite mixing, the droplets generally remained separate from each other.
  • Vaheat coverslips Interherence, Erlangen, Germany
  • the coverslips coated with TiO 2 acting as a resistor, can rapidly reach and maintain specific temperatures.
  • the coverslips have embedded sensors to provide feedback control of their temperature (visible in the Figure 14A image towards the upper right).
  • the droplets were sandwiched between two coverslips by placing the droplet suspension on top of a Vaheat coverslip, and then placing a regular glass coverslip on top of the droplet suspension. This setup formed a monolayer of droplets due to surface tension.
  • a hydrophobic coating (Gel-Slick, Thermo Fisher Scientific, Waltham, MA) was applied to both coverslips.
  • the boundaries of the droplets were then adjusted (shrunk) to focus on the droplet cores, and the larger droplets were excluded because they likely arose from the merger of more than one droplet.
  • the selected single droplets are indicated by polygons drawn on the image in Figure 14A and their labels are identified by numbers in this image.
  • the mean fluorescence intensity at each temperature in the core of each selected droplet (within the polygons) was then noted, creating a table of fluorescence for each droplet as a function of temperature.
  • the resulting fluorescence vs. temperature curves were then normalized to the same value at 72°C, because all of the hybrids can be assumed to be fully dissociated at this temperature, resulting in an identical fluorescence intensity (see Figure 5).
  • targets can also be derived from amplified TCR mRNAs, or genes in single T cells.
  • the melt profile of each droplet will indicate the TCR arrangement of the encapsulated T cell, and the ensemble of profiles would represent the sample's TCR repertoire.
  • 238 droplets were imaged over time in a single field of view. To increase the number of imaged droplets, a larger region encompassing more droplets can be scanned. This is routinely accomplished by moving the microscope stage in a programmed manner while capturing images. These images, or “tiles,” can then be stitched together into a mosaic that includes a greater number of droplets.
  • a third strategy involves imaging the droplets within a three-dimensional volume, such as a solution inside a test tube. Imaging droplet fluorescence in three dimensions within test tubes has been previously described utilizing light-sheet fluorescence imaging (Liao P et al. Three-dimensional digital PCR through light-sheet imaging of optically cleared emulsion. Proc Natl Acad Sci U S A (2020) 117:25628-25633.). This technique has the potential to rapidly observe up to half a million droplets while changing the temperature and thereby determine the melt profiles of DNA molecules encapsulated therein.
  • the present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des acides nucléiques, des réactifs et des procédés d'établissement de profils de répertoires de récepteurs cellulaires. Le procédé divulgué permet de distinguer quels agencements de récepteurs immunitaires sont présents dans des cellules individuelles parmi les millions d'agencements de récepteurs immunitaires possibles. En outre, le procédé peut rendre compte de l'agencement des récepteurs immunitaires présents dans chacune des 100 000 cellules ou plus dans un seul dosage, définissant ainsi le répertoire immunitaire d'un individu. Notamment, le procédé présenté est applicable à l'établissement de profils des répertoires de divers récepteurs présentant des réarrangements somatiques, y compris celui des récepteurs des lymphocytes T ou des récepteurs des lymphocytes B.
PCT/US2024/045631 2023-09-14 2024-09-06 Procédés d'établissement de profils de répertoires immunitaires Pending WO2025058951A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363582589P 2023-09-14 2023-09-14
US63/582,589 2023-09-14

Publications (1)

Publication Number Publication Date
WO2025058951A1 true WO2025058951A1 (fr) 2025-03-20

Family

ID=95021812

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/045631 Pending WO2025058951A1 (fr) 2023-09-14 2024-09-06 Procédés d'établissement de profils de répertoires immunitaires

Country Status (1)

Country Link
WO (1) WO2025058951A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008026927A2 (fr) * 2006-08-30 2008-03-06 Academisch Medisch Centrum Procédé d'affichage des répertoires des récepteurs des lymphocytes t et b
US20130029855A1 (en) * 2011-07-25 2013-01-31 Bioinventors & Entrepreneurs Network, Llc Sieving of Nucleic Acid Samples
US20180044726A1 (en) * 2012-06-15 2018-02-15 Board Of Regents, The University Of Texas System High throughput sequencing of multiple transcripts
US20190367585A1 (en) * 2016-04-12 2019-12-05 Medimmune, Llc Immune repertoire mining

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008026927A2 (fr) * 2006-08-30 2008-03-06 Academisch Medisch Centrum Procédé d'affichage des répertoires des récepteurs des lymphocytes t et b
US20130029855A1 (en) * 2011-07-25 2013-01-31 Bioinventors & Entrepreneurs Network, Llc Sieving of Nucleic Acid Samples
US20180044726A1 (en) * 2012-06-15 2018-02-15 Board Of Regents, The University Of Texas System High throughput sequencing of multiple transcripts
US20190367585A1 (en) * 2016-04-12 2019-12-05 Medimmune, Llc Immune repertoire mining

Similar Documents

Publication Publication Date Title
JP7220247B2 (ja) デジタル検体分析
US7618778B2 (en) Producing, cataloging and classifying sequence tags
US11125752B2 (en) Method for rapid accurate dispensing, visualization and analysis of single cells
DK2970959T3 (en) METHODS AND COMPOSITIONS FOR MARKING AND ANALYSIS OF SAMPLES
CN100494395C (zh) 通过同时探查和酶介导检测的多态性基因座多重分析
CN105408495B (zh) 通过茎环结构阻断3’dna末端的聚合酶延伸的方法
CN103348017B (zh) 使用检测剂、探针和抑制剂检测核酸靶标
JP2005509442A (ja) 免疫遺伝子レパートリーのプロファイリング
EP3524693A1 (fr) Analyse d'analytes numérique
WO2002061122A2 (fr) Nouvelle methode de determination du genotype
JP5060945B2 (ja) 癌診断のためのオリゴヌクレオチド
US20100086918A1 (en) Methods for High Sensitivity Detection of Genetic Polymorphisms
US10975440B2 (en) Experimentally validated sets of gene specific primers for use in multiplex applications
JP6846832B2 (ja) 一細胞の非破壊的計測情報とゲノム関連情報とを一体的に検出する方法
US11655510B2 (en) Experimentally validated sets of gene specific primers for use in multiplex applications
WO2025058951A1 (fr) Procédés d'établissement de profils de répertoires immunitaires
US20090061440A1 (en) Method for amplifying plural nucleic acid sequences for discrimination
DE102007031137A1 (de) Verfahren und Sonden/Primärsystem zum "real time" Nachweis eines Nukleinsäuretargets
US20040086867A1 (en) Method for detecting nucleic acid
CN1147591C (zh) 聚合酶链式反应基因芯片
CN120225460A (zh) 测序方法
HK1205534A1 (en) Method of dna detection and quantification by single-molecule hybridization and manipulation
HK1205534B (en) Method of dna detection and quantification by single-molecule hybridization and manipulation

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: 24866109

Country of ref document: EP

Kind code of ref document: A1