Use of homologous recombinase to improve efficiency and sensitivity of single cell assays

Abstract

Methods of reducing bead aggregation and increasing the specificity of nucleic acid hybridization using non-specific nucleic acid binding proteins are described.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 352,975, filed on June 16, 2022, which is incorporated by reference for all purposes. Background Art

[0003] Achieving high sensitivity in single-cell RNA applications involves efficient capture of RNA, conversion to cDNA, and generation of sequencing libraries. The single-cell assay input has relatively few transcripts to capture, so efficiency loss in any of these steps results in loss of gene coverage and diversity. For example, several low-transcript genes have as few as 5-12 copies per cell. Summary of the invention

[0004] In some embodiments, a method of introducing a bead of linked oligonucleotides into a partition is provided. In some embodiments, the method comprises

[0005] providing a bulk solution comprising (i) a plurality of beads of said linked oligonucleotides and (ii) a non-sequence-specific nucleic acid binding protein in an amount sufficient to reduce aggregation of said beads; and

[0006] Introducing the beads into the partitions. In some embodiments, the number of beads in the partitions is less than 1, 2, 3, 4, or 5 beads per partition on average.

[0007] In some embodiments, the partitions are microwells or droplets.

[0008] In some embodiments, at least a majority of the oligonucleotides coated on the beads have the same sequence, and oligonucleotides on different beads can be distinguished by a barcode sequence on the oligonucleotides.

[0009] In some embodiments, the oligonucleotide includes a free 3' end including at least 3, 4, 5, or 6 consecutive thymine nucleotides forming a polythymidylic acid (poly-T) sequence.

[0010] In some embodiments, during the introduction, the partition includes a single cell in each partition. In some embodiments, after the introduction, the cells in the partition are cracked and the oligonucleotides in the partition are hybridized with the target nucleic acid from the cell. In some embodiments, the method also includes adding an additional amount of non-sequence-specific nucleic acid binding protein to the partition before hybridization. In some embodiments, the oligonucleotide is connected to the bead before hybridization. In some embodiments, the oligonucleotide is released from the bead before hybridization. In some embodiments, the method also includes utilizing a polymerase to extend the oligonucleotide in a template-specific manner to connect the reverse complementary sequence of the target nucleic acid to the oligonucleotide.

[0011] In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is DNA.

[0012] In some embodiments, the non-sequence-specific nucleic acid binding protein is selected from the group consisting of RecA, RecO, RecN, RadA, Rad51, Rad52, and UvsX.

[0013] A solution is also provided, comprising (i) a plurality of beads connected to oligonucleotides and (ii) a non-sequence-specific nucleic acid binding protein in an amount sufficient to reduce aggregation of the beads. In some embodiments, the oligonucleotide comprises a free 3' end, the free 3' end comprising at least 6 consecutive thymine nucleotides, the thymine nucleotides forming a poly-thymidylate sequence. In some embodiments, the non-sequence-specific nucleic acid binding protein is selected from the group consisting of: RecA, RecO, RecN, RadA, Rad51, Rad52 and UvsX.

[0014] A method for introducing a bead of linked oligonucleotides into a partition is also provided. In some embodiments, the method comprises providing a plurality of partitions, wherein the partitions comprise (i) beads of linked oligonucleotides and (ii) a non-sequence-specific nucleic acid binding protein, and (iii) a target nucleic acid; and in the presence of the non-sequence-specific nucleic acid binding protein, hybridizing the oligonucleotide to the target nucleic acid.

[0015] In some embodiments, the number of beads in a partition is less than 1, 2, 3, 4, or 5 beads per partition on average.

[0016] In some embodiments, the method comprises releasing the oligonucleotide from the bead prior to hybridization.

[0017] In some embodiments, the oligonucleotide includes a free 3' end that includes at least 3, 4, 5, or 6 consecutive thymine nucleotides that form a poly-thymidylate sequence.

[0018] In some embodiments, at least a majority of the oligonucleotides attached to the beads have the same sequence, and oligonucleotides on different beads can be distinguished by a barcode sequence on the oligonucleotide.

[0019] In some embodiments, the partitions are microwells or droplets.

[0020] In some embodiments, the non-sequence-specific nucleic acid binding protein is selected from the group consisting of RecA, RecO, RecN, RadA, Rad51, Rad52, and UvsX.

[0021] Also provided is a reaction mixture comprising (i) beads to which oligonucleotides are attached and (ii) a non-sequence-specific nucleic acid binding protein, and (iii) a target nucleic acid.

[0022] In some embodiments, the non-sequence-specific nucleic acid binding protein is selected from the group consisting of RecA, RecO, RecN, RadA, Rad51, Rad52, and UvsX.

[0023] Also provided are multiple partitions, wherein the partitions contain a reaction mixture as described above or elsewhere herein, wherein at least a majority of the oligonucleotides attached to the beads in the partitions have the same sequence, and the oligonucleotides on different beads in different partitions can be distinguished by a barcode sequence on the oligonucleotides. In some embodiments, the partitions are microwells or droplets.

[0024] A method for detecting nucleic acids in a microfluidic channel is also provided. In some embodiments, the method comprises flowing a nucleic acid sample through an immobilized oligonucleotide-linked bead in a microfluidic channel in the presence of a non-sequence-specific nucleic acid binding protein under conditions sufficient to allow the oligonucleotide to specifically hybridize with a target nucleic acid in the nucleic acid sample; and detecting hybridization of the target nucleic acid with the oligonucleotide. In some embodiments, the oligonucleotide-linked bead is immobilized in the microfluidic channel by optical tweezers.

[0025] definition

[0026] Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally performed according to conventional methods and various general references in the art (generally referring to, Sambrook et al. "Molecular Cloning: A Laboratory Manual (MOLECULARCLONING: A LABORATORY MANUAL)", 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the document is incorporated herein by reference), and the conventional methods and various general references are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis described below are well-known and commonly used in the art.

[0027] As used herein, the terms "a", "an" or "the" encompass not only aspects having one member, but also aspects having more than one member. For example, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a bead" includes a plurality of such beads, and reference to "the sequence" includes reference to one or more sequences known to those skilled in the art, etc.

[0028] An "oligonucleotide" is a polynucleotide. Typically, an oligonucleotide will have less than 250 nucleotides, in some embodiments, 4-200, such as 10-150 nucleotides.

[0029] A "clonal" copy of a polynucleotide means that the sequence of the copies is identical. In some embodiments, there are at least 100, 1000, 10 4 or more cloned copies.

[0030] The term "amplification reaction" refers to any in vitro means of amplifying copies of a target sequence of a nucleic acid in a linear or exponential manner. Such methods include, but are not limited to, polymerase chain reaction (PCR); DNA ligase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., ed., 1990)) (LCR); QBeta RNA replicase and RNA transcription-based amplification reactions (e.g., amplification involving RNA polymerization initiated by T7, T3 or SP6), such as transcription amplification system (TAS), nucleic acid sequence-based amplification (NASBA) and self-sustained sequence replication (3SR); isothermal amplification reactions (e.g., single primer isothermal amplification (SPIA)); and other techniques known to those skilled in the art.

[0031] "Amplification" refers to the step of placing a solution under conditions sufficient to amplify a polynucleotide if all components of the reaction are intact. The components of an amplification reaction include, for example, primers, a polynucleotide template, a polymerase, nucleotides, etc. The term "amplification" generally refers to an "exponential" increase in a target nucleic acid. However, as used herein, "amplification" may also refer to a linear increase in the number of a selected target sequence of a nucleic acid, such as obtained by cycle sequencing or linear amplification. In an exemplary embodiment, amplification refers to PCR amplification using an amplification primer and a first amplification primer.

[0032] The term "amplification reaction mixture" refers to an aqueous solution comprising various reagents used to amplify a target nucleic acid. These include enzymes, aqueous buffers, salts, amplification primers, target nucleic acids, and nucleoside triphosphates. The amplification reaction mixture may further include stabilizers and other additives to optimize efficiency and specificity. Depending on the context, the mixture may be a complete or incomplete amplification reaction mixture.

[0033] "Polymerase chain reaction" or "PCR" refers to a method for geometrically amplifying a specific segment or subsequence of a target double-stranded DNA. PCR is well known to those skilled in the art; see, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202; and "PCR Protocols: A Guide to Methods and Applications," ed. Innis et al., 1990. Exemplary PCR reaction conditions typically include two or three-step cycles. A two-step cycle has a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step followed by a separate extension step.

[0034] "Primer" refers to a polynucleotide sequence that hybridizes with a sequence on a target nucleic acid and serves as a starting point for nucleic acid synthesis. Primers can have various lengths, and the length is generally less than 50 nucleotides, for example, 12 to 30 nucleotides in length. The length and sequence of primers used for PCR can be designed based on principles known to those skilled in the art, see, for example, Innis et al., supra. Primers can be chimeras of DNA, RNA, or DNA and RNA portions. In some cases, primers can include one or more modified or non-natural nucleotide bases. In some cases, primers are labeled.

[0035] Nucleic acid or a part thereof "hybridizes" with another nucleic acid under certain conditions, so that non-specific hybridization at a temperature limited in a physiological buffer (e.g., pH 6-9, 25-150mM chloride salt) is minimized. In some cases, nucleic acid or a part thereof hybridizes with a conserved sequence common to a group of target nucleic acids. In some cases, if there are at least about 6, 8, 10, 12, 14, 16 or 18 continuous complementary nucleotides, comprising "universal" nucleotides complementary to more than one nucleotide partner, a primer or a part thereof can hybridize with a primer binding site. Alternatively, if there are less than 1 or 2 complementary mispairings on at least about 12, 14, 16 or 18 continuous complementary nucleotides, a primer or a part thereof can hybridize with a primer binding site. In certain embodiments, the temperature at which the definition of specific hybridization occurs is room temperature. In certain embodiments, the temperature at which the definition of specific hybridization occurs is higher than room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37° C., 40° C., 42° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C. In some embodiments, the defined temperature at which specific hybridization occurs is 37° C., 40° C., 42° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.

[0036] A "template" refers to a polynucleotide sequence that includes a polynucleotide to be amplified, flanked by a primer hybridization site or a pair of primer hybridization sites. Thus, a "target template" includes a target polynucleotide sequence adjacent to at least one hybridization site for a primer. In some cases, a "target template" includes a target polynucleotide sequence flanked by hybridization sites for a "forward" primer and a "reverse" primer.

[0037] As used herein, "nucleic acid" means DNA, RNA, single-stranded, double-stranded or more highly aggregated hybridization motifs and any chemical modifications thereof. Modifications include, but are not limited to, modifications that provide chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interactions, attachment points, and functionality for nucleic acid ligand bases or for the entire nucleic acid ligand. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiesterase group modifications (e.g., phosphorothioates, methylphosphonates), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitutions of 4-thiouridine, substitutions of 5-bromine or 5-iodo-uracil; backbone modifications, methylation, unusual base pairing combinations, such as isobases, isocytidines, and isoguanidines, etc. Nucleic acids may also include non-natural bases, such as nitroindole. Modifications may also include 3' and 5' modifications, which include, but are not limited to, end-capping with fluorophores (e.g., quantum dots) or another portion.

[0038] "Polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides (e.g., DNA and / or RNA). The term encompasses both full-length polypeptides and domains having polymerase activity. DNA polymerases are well known to those skilled in the art and include, but are not limited to, DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. Additional examples of commercially available polymerases include, but are not limited to, Klenow fragment (New England Biolabs, Inc., Ill.), and the like. Co., Ltd.), Taq DNA polymerase (QIAGEN), 9°N TM DNA polymerase (New England Co., Ltd.), Deep Vent TM DNA polymerase (New England Co., Ltd.), Manta DNA polymerase Bst DNA polymerase (New England Inc.) and phi29 DNA polymerase (New England Biolabs, Inc.) Company Limited).

[0039] Polymerases include both DNA-dependent polymerases and RNA-dependent polymerases, such as reverse transcriptases. It is known that there are at least five DNA-dependent DNA polymerase families, although most belong to A, B and C families. Other types of DNA polymerases include bacteriophage polymerases. Similarly, RNA polymerases generally include eukaryotic RNA polymerases I, II and III, as well as bacterial RNA polymerases and bacteriophage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

[0040] As used herein, the term "partition" or "partitioned" refers to separating a sample into multiple parts or "partitions". Partitions are typically physical, so that a sample in one partition does not or substantially does not mix with a sample in an adjacent partition. Partitions can be solid or fluid. In some embodiments, the partitions are solid partitions, e.g., microchannels. In some embodiments, the partitions are fluid partitions, e.g., droplets. In some embodiments, the fluid partitions (e.g., droplets) are mixtures of immiscible fluids (e.g., water and oil). In some embodiments, the fluid partitions (e.g., droplets) are aqueous droplets surrounded by an immiscible carrier fluid (e.g., oil). Before the solution is dispensed, the solution is referred to as a "bulk" solution.

[0041] As used herein, a "barcode" is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, 12, 15, 20, 50 or 75 or 100 or more nucleotides in length) that identifies a molecule conjugated thereto or from a partition from which it originates. Barcodes can be used, for example, to identify molecules derived from partitions, beads or spots, such as those sequenced later from bulk reactions. Such barcodes may be unique to the partitions, beads or spots compared to barcodes present in other partitions, beads or spots. For example, a partition containing a target RNA from a single cell can be subjected to reverse transcription conditions using primers containing different partition-specific barcode sequences in each partition, thereby incorporating a copy of a unique "cell barcode" (because different cells are in different partitions, and each partition has a unique partition-specific barcode) into the reverse transcribed nucleic acid of each partition. Therefore, due to the unique "cell barcode", nucleic acids from each cell can be distinguished from nucleic acids from other cells. In some embodiments described herein, the barcodes described herein uniquely identify the molecule to which they are conjugated, i.e., the barcodes serve as unique molecular identifiers (UMIs). The length of a possible barcode sequence determines how many unique samples can be distinguished. For example, depending on degeneracy, a 1 nucleotide barcode can distinguish 4 or fewer different partitions; a 4 nucleotide barcode can distinguish 4 4 or 256 partitions or less; a 6-nucleotide barcode can distinguish 4096 different partitions or less; and an 8-nucleotide barcode can index 65,536 different partitions or less.

[0042] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0043] "Conservatively modified variants" are applicable to both amino acid sequences and nucleic acid sequences. With respect to specific nucleic acid sequences, "conservatively modified variants" refer to those nucleic acid sequences that encode the same or substantially the same amino acid sequence, or when the nucleic acid does not encode an amino acid sequence, it refers to substantially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where the codon is designated as alanine, the codon can be changed to any one of the corresponding codons described without changing the encoded polypeptide. This nucleic acid variation is a "silent variation", which is a kind of conservatively modified variation. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The technician will recognize that each codon in the nucleic acid (except AUG and TGG, AUG is usually the only codon for methionine, and TGG is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of the nucleic acid encoding a polypeptide is implicit in each described sequence.

[0044] With respect to amino acid sequences, the skilled artisan will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete a single amino acid or a small number of amino acids in the coded sequence are "conservatively modified variants," wherein the alteration causes the amino acid to be substituted with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues and alleles of the present invention.

[0045] The following eight groups each contain amino acids that are conservative substitutions for each other:

[0046] 1) Alanine (A), glycine (G);

[0047] 2) Aspartic acid (D), glutamic acid (E);

[0048] 3) Asparagine (N), glutamine (Q);

[0049] 4) Arginine (R), Lysine (K);

[0050] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0051] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0052] 7) serine (S), threonine (T); and

[0053] 8) Cysteine ​​(C), Methionine (M)

[0054] (See, eg, Creighton, Proteins, WH Freeman and Co., New York (1984)).

[0055] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides likewise may be referred to by their commonly accepted single-letter codes.

[0056] As used herein, in the case of describing two or more polynucleotides or amino acid sequences, the term "identical" or "identity" percentage refers to identical two or more sequences or a specified subsequence. When comparing and aligning in a comparison window or a specified region to obtain maximum correspondence, as measured using a sequence comparison algorithm or by manually aligning and visual inspection (when a specific region is not specified), two sequences of "substantially identical" have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. With regard to polynucleotide sequences, this definition also refers to the complementary sequence of a test sequence. With regard to amino acid sequences, in some cases, identity is present in a region where a length is at least about 50 amino acids or nucleotides, or more preferably a length is in a region where a length is 75-100 amino acids or nucleotides.

[0057] For sequence comparison, usually a sequence serves as a reference sequence to compare with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters. For the sequence comparison of nucleic acids and proteins, BLAST 2.0 algorithm and the default parameters discussed below are used.

[0058] As used herein, a "comparison window" includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence having the same number of contiguous positions after optimal alignment.

[0059] The algorithm used to determine the percentage of sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analysis is publicly available at the National Center for Biotechnology Information website ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing the initial neighborhood word hits. Then, word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0) are used to calculate the cumulative score. For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word match in each direction ceases when: the cumulative alignment score decreases by the amount X from its maximum achieved; the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0060] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. DETAILED DESCRIPTION

[0061] It has been determined that one can use nonspecific nucleic acid binding proteins to coat certain polynucleotides in various manipulations of nucleic acid solutions, such as, but not limited to, partitioning and hybridization, which can improve the specificity of the reaction and reduce undesirable aggregation of molecular biology solution components. This technology can be applied to both bulk and compartmentalized reactions, such as those performed in test tubes, droplets, or microwells. In addition, improving homologous interactions with the aid of nonspecific nucleic acid binding proteins can increase the sensitivity and efficiency of oligonucleotide-based detection assays and PCR.

[0062] It is not intended to limit the scope of the present invention, it is believed that the inclusion of non-sequence-specific nucleic acid binding proteins (e.g., recombination and DNA repair DNA / RNA binding proteins) in a solution with nucleic acids reduces the non-specific intramolecular and intermolecular interactions of oligonucleotides connected in solution or on beads, and can be used to increase the capture of target-specific sequences via oligonucleotide hybridization. The inclusion of non-specific nucleic acid binding proteins in a solution with beads also reduces the aggregation of beads. Since beads connected to oligonucleotides are usually used to deliver oligonucleotides to partitions, beads comprising non-sequence-specific nucleic acid binding proteins and connected oligonucleotides allow for improved distribution by, for example, reducing aggregation, otherwise aggregation can result in a higher level of multiple beads in a partition.

[0063] A variety of non-sequence specific nucleic acid binding proteins are known, and these non-sequence specific nucleic acid binding proteins can be used in the methods and compositions described herein. In this case, "non-sequence specificity" refers to the ability of proteins to bind to nucleic acids independently of nucleic acid sequences. Although certain binding changes may occur between sequences, in general, non-sequence specific binding proteins will bind to many different nucleic acid sequences with substantially similar affinity. This is in contrast to, for example, transcription factors or sequence-specific restriction enzymes, which target specific sequences or small amounts of sequences with an affinity that is significantly stronger than the affinity for other non-target sequences (e.g., 100 times or more). The benefit of non-sequence specific nucleic acid binding proteins is that many or most sequences within the nucleic acid molecule will be combined to a certain extent, so that the effects described herein occur in a measurable manner.

[0064] In some embodiments, the non-sequence specific nucleic acid binding protein is selected from nucleic acid (e.g., DNA) recombination proteins and / or DNA repair proteins. Exemplary non-specific nucleic acid binding proteins may include, for example, RecA, RecO, RecN, RadA, Rad51, Rad52, and UvsX. It should be understood that the orthologous proteins of each of these proteins can be identified from many species and used as described herein.

[0065] Exemplary RecA proteins may include, but are not limited to, E. coli or Thermus aquaticus RecA, such as described by Uniprot Accession Nos. P0A7G6 or P48296, respectively, or a RecA protein substantially identical to:

[0066] MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGSLSLDIALGAGGLPMGRIVEIYGPESSGKTTITLQVIAAAQREGKTCAFIDAEHALDPIYARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDSHMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASVRLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVKEKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSVDDSEGVAETNEDF or MEENKRKSLENALKTIEKEF GKGAVMRLGE MPKLQVDVIPTGSLGLDLAL GIGGIPRGRV TEIFGPESGG KTTLALTIIAQAQKGGGVAAFVDAEHALDPLYAKKLGVDV QELLVSQPDT GEQALEIVELLARSGAVDVI VVDSVAAIVPKAEIEGEMGD QHVGLQARLMSQALRKLTAV LSKSNTAAIF INQVREKVGV MYGNPETTPGGRALKFYSSVRLDVRKSGQP IKVGNEAVGI KVKVKVVKNKLAPPFREAEL EIYFGRGLDPVMDLVNVAVAAGVIEKAGSWFSYGEHRLGQ. See also Del Val et al., Biochem Soc Trans. October 31, 2019; 47(5): 1511-153.

[0067] Exemplary RecO proteins may include, but are not limited to, E. coli RecO, such as described by Uniprot Accession No. P0A7H3, or a RecO protein substantially identical to: MEGWQRAFVLHSRPWSETSLMLDVFTEESGRVRLVAKGARSKRSTLKGAL QPFTPLLLRFGGRGEVKTLRSAEAVSLALPLSGITLYSGLYINELLSRVLEYETRFSELFFDYLHCIQSLAGVTGTPEPALRRFELALLGHLGYGVNFTHCAGSGEPVDDTMTYRYREEKGFIASVVIDNKTFTGRQLKALNAREFPDADTLRAAKRFTRMALKPYLGGKPLKSRELFRQFMPKRTVKTHYE.

[0068] Exemplary Rad51 proteins may include, but are not limited to, human Rad51, such as described by Uniprot Accession No. Q06609, or a Rad51 protein substantially identical to: GSHMAMQMQL

[0069]

[0070] Exemplary Rad52 proteins may include, but are not limited to, human Rad52, such as described by Uniprot Accession No. P43351, or a Rad52 protein substantially identical to:

[0071] MSGTEAILGGRDSHPAAGGGSVLCFGQCQYTAEEYQAIQKALRQRLGPEYISSRMAGGGQKVCYIEGHRVINLANEMFGYNGWAHSITQQNVDFVDLNNGKFYVGVCAFVRVQLKDGSYHEDVGYGVSEGLKSKALSLEKARKEAVTDGLKRALRSFGNALGNCILDKDYLRSLNKLPRQLPLEVDLTKAKRQDLEPSV EEARYNSCRPNMALGHPQLQQVTSPSRPSHAVIPADQDCSSRSLSSSAVESEATHQRKLRQKQLQQQFRERMEKQQVRVSTPSAEKSEAAPPAPPVTHSTPVTVSEPLLEKDFLAGVTQELIKTLEDNSEKWAVTPDAGDGVVKPSSRADPAQTSDTLALNNQMVTQNRTPHSVCHQKPQAKSGSWDLQTYSADQRTTGN WESHRKSQDMKKRKYDPS. See also McDevitt et al., Nature Communications, Vol. 9, Article No. 1091 (2018).

[0072] Exemplary UvsX proteins may include, but are not limited to, bacteriophage T4 UvsX described under Uniprot Accession No. P04529, or a UvsX protein substantially identical to:

[0073]

[0074] In certain embodiments, two or more different (different amino acid sequences) non-sequence specific nucleic acid proteins can be used. For example, 2, 3, 4, 5 or more different non-sequence specific nucleic acid proteins can be used in the methods described herein. In certain embodiments, using two or more different non-sequence specific nucleic acid proteins can allow for improved results.

[0075] In some embodiments, one or two or more non-sequence-specific nucleic acid binding proteins are mixed with a plurality of oligonucleotide-linked beads in solution. This can be done in bulk solution, i.e., before dispensing the solution, thereby allowing the oligonucleotides in the oligonucleotide-linked beads to bind to the non-sequence-specific nucleic acid binding proteins, thereby reducing or preventing aggregation between the beads.

[0076] The beads of exemplary oligonucleotides are connected, for example, comprise hydrogel beads. The beads connected to the cloned copies of oligonucleotides are generally used for bead-specific (e.g., partition-specific) barcodes and other barcodes to be delivered to partitions. Depending on the type of, for example, partitions, various methods can be used for inserting beads into partitions. However, in general, a goal of inserting beads into partitions for connecting oligonucleotides is to realize that each partition has a single bead. However, the beads of oligonucleotides may have certain affinity to each other, resulting in the aggregation of beads, so that bead aggregates are delivered to some partitions, rather than single bead delivery. By initially combining the mixture of beads of oligonucleotides with non-sequence-specific nucleic acid binding proteins, the aggregation of beads of oligonucleotides will be reduced, thereby allowing beads to be delivered to partitions.

[0077] The hydrogel beads can be made of various materials. Exemplary hydrogel beads can be, for example, agarose hydrogel beads or polyacrylamide hydrogel beads. In other embodiments, the hydrogel includes, for example, polystyrene, silica, polymethyl methacrylate, alginate, polyethylene glycol (PEG), nylon, and / or other polymers, with or without crosslinking. Other hydrogels include, but are not limited to, those described in, for example, U.S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U.S. Patent Application Nos. 2002 / 0,009,591; 2013 / 0,022,569; 2013 / 0,034,592; and International Patent Publication Nos. WO / 1997 / 030092; and WO / 2001 / 049240. The beads can be magnetic or paramagnetic as desired.

[0078] As required, the oligonucleotide can be connected to the beads. The solid support surface of the beads can be modified to include a joint for attaching the barcode oligonucleotide. The joint can include a cleavable portion. Non-limiting examples of cleavable portions include disulfide bonds, dioxyuridine moieties, and restriction enzyme recognition sites. The method of connecting the oligonucleotide to the beads is described in, for example, WO2015 / 200541. In certain embodiments, the oligonucleotide configured to connect the hydrogel beads to the barcode is covalently linked to the hydrogel. Many methods for covalently linking an oligonucleotide to one or more hydrogel matrices are known in the art. As an example, aldehyde-derivatized agarose can be covalently linked to the 5′-amine group of a synthetic oligonucleotide.

[0079] The beads of the oligonucleotides connected to which the non-sequence-specific nucleic acid binding protein is bound can be distributed to the partitions by any available method. For example, the partitions can be optionally preformed with other reagents and optionally target nucleic acids from biological samples, and beads and non-covalently linked oligonucleotides can be injected or otherwise introduced into the partitions. The methods and compositions for delivering reagents to one or more partitions include microfluidic methods known in the art; droplets or microcapsules merge, coalesce, merge, rupture or degrade (for example, as described in US2015 / 0027,892; US2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (for example, as described in WO 2010 / 151,776); and combinations thereof.

[0080] In other embodiments, for example, where the partitions are droplets, droplets in the form of an emulsion can be formed with an immiscible fluid such as oil, whereby a bulk solution is formed into droplets containing beads and non-covalently attached oligonucleotides, optionally other reagents and / or sample nucleic acids. Methods of emulsion formation are described, for example, in published patent applications WO 2011 / 109546 and WO 2012 / 061444.

[0081] In some embodiments, the distribution of beads into partitions (e.g., droplets) can be determined by a Poisson distribution. Depending on the end use, the average number of beads per partition can be less than 1 (e.g., 0.2-0.9), equal to 1 or greater than 1 (e.g., 1-3 or greater, such as less than 5). In some embodiments, it is desirable to avoid having multiple beads in a partition, and in these cases, many partitions may be left empty so that most partitions containing beads contain only one bead. In other embodiments, for example, where a deconvolution method can be used to decrypt sequencing results in which multiple beads appear in a single partition, each partition can be loaded with more beads on average, and then deconvolution is used to parse the sequencing results. See, for example, PCT / US2017 / 012618, PCT / US2019 / 015638, PCT / US2020 / 36699. However, if the beads are aggregated less, it will be beneficial even if more than one bead is inserted in a partition.

[0082] Any type of partition can be used with the methods and compositions described herein. In certain embodiments, beads can be inserted into partitions (e.g., droplets or holes). In certain embodiments, beads are encapsulated in aqueous droplets in water-in-oil emulsions. For example, methods and compositions for partitioning samples are described in published patent applications WO 2010 / 036,352, US2010 / 0173,394, US2011 / 0092,373 and US2011 / 0092,376, each of which is incorporated herein by reference in its entirety. Multiple mixture partitions can be located in multiple emulsion droplets or multiple micropores, etc. Partitions can be picopores, nanopores or micropores. In certain embodiments, there are at least, for example, 100,000 holes or 200,000 holes, for example, 100,000-500,000 holes. The volume capacity of exemplary holes can be, for example, 10-50 picoliters. Exemplary wells include those described in U.S. Patent Publication No. US2021 / 0283608. The mixture partition may be a picochamber, a nanochamber, or a microreaction chamber, such as a picocapsule, a nanocapsule, or a microcapsule. The mixture partition may be a picochannel, a nanochannel, or a microchannel. The mixture partition may be a droplet, for example, an emulsion droplet.

[0083] In addition to improving the distribution of beads in partitions by reducing aggregation, another advantageous use of non-sequence-specific nucleic acid binding proteins is to improve nucleic acid-specific nucleic acid hybridization and reduce non-specific hybridization. Therefore, non-sequence-specific nucleic acid binding proteins can be included in any nucleic acid reaction mixture to increase the sensitivity and efficiency of oligonucleotide-based hybridization. Therefore, for example, any type of nucleic acid hybridization assay can be improved by including a sufficient amount of non-specific nucleic acid binding proteins in the reaction mixture of the method, including but not limited to nucleic acid amplification methods, including but not limited to PCR.

[0084] Therefore, in some embodiments, under conditions of inducing hybridization, a non-sequence-specific nucleic acid binding protein is included in a mixture including an oligonucleotide and a target nucleic acid, and then the specific hybridization between the oligonucleotide and the target nucleic acid (if present) is detected. The amount of the non-specific nucleic acid binding protein included can be sufficient to improve the specificity of complementary nucleic acids (e.g., oligonucleotides and target nucleic acids), optionally by reducing non-sequence-specific nucleic acid interactions, such as non-100% complementary nucleic acids to improve specificity. As required, the improved specificity of hybridization of complementary nucleic acids can be determined. In one embodiment, specificity can be determined by hybridizing an oligonucleotide connected to a bead with a mixture of completely and partially complementary nucleic acids in the presence and absence of one or more non-sequence-specific nucleic acid binding proteins, removing non-hybridized nucleic acids from the bead, and detecting the amount of partially complementary nucleic acids that remain associated with the bead. This can involve, for example, precipitating beads in a hybridization reaction, washing off non-hybridized nucleic acids, then eluting hybridized nucleic acids and determining the amount of non-completely complementary nucleic acids via, for example, qPCR. Compared to a control that is otherwise identical but lacks non-sequence-specific nucleic acid binding proteins, containing a sufficient amount of non-sequence-specific nucleic acid binding proteins will result in less hybridization of non-completely complementary nucleic acids.

[0085] Any form for detecting nucleic acid hybridization can be used. In certain embodiments, the target nucleic acid can be optionally amplified in a partition, and then the amplified target nucleic acid can be detected, preferably so that the presence or absence of an amplified product can be optically detected. Amplification can be carried out under isothermal or thermal cycle conditions. For example, amplification may damage reverse transcription (RT), RT-PCR, loop-mediated isothermal amplification (LAMP) (LAMP is isothermal), RT-LAMP or bridge amplification.

[0086] As required, the target nucleic acid can be any nucleic acid. For example, the nucleic acid can be a target nucleic acid from a biological sample. The biological sample can be obtained from any biological organism, for example, an animal, a plant, a fungus, a pathogen (for example, a bacterium or a virus) or any other organism. In some embodiments, the biological sample is from an animal, such as a mammal (for example, a human or non-human primate, a cow, a horse, a pig, a sheep, a cat, a dog, a mouse or a rat), a bird (for example, a chicken) or a fish. The biological sample can be any tissue or body fluid obtained from a biological organism, for example, blood, a blood fraction or a blood product (for example, serum, plasma, platelets, red blood cells, etc.), sputum or saliva, a tissue (for example, kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage or bone tissue); cultured cells, such as primary cultures, explants and transformed cells, stem cells, feces, urine, etc. In some embodiments, the sample is a sample comprising a cell. In some embodiments, the sample is a single cell sample.

[0087] In some embodiments, the target nucleic acid is RNA. RNA can be reverse transcribed by adding appropriate reverse transcription reagents (e.g., reverse transcriptase, nucleotides, one or more primers, the primers are optionally primers comprising polythymidylic acid 3' ends) and sufficient amounts of non-sequence-specific nucleic acid binding proteins described herein, and RNA is used as a template to extend the primer to form a first-strand cDNA molecule. As another possible step, the first-strand cDNA can be converted into double-stranded cDNA by second-strand synthesis (e.g., by providing appropriate reagents, such as appropriate primers and DNA polymerase).

[0088] Optionally, hybridization or detection of hybridization or both can occur in partitions, allowing, for example, a "digital" signal, where each partition produces a detectable signal if an oligonucleotide probe or primer hybridizes to a target nucleic acid in the partition. Quantification of hybridization can be determined by quantifying the number of partitions that produce a signal, optionally as a ratio of the total number of partitions or the total number of oligonucleotide primers or probes contained.

[0089] In other embodiments, one or more non-sequence-specific nucleic acid binding proteins are included in the hybridization performed in the microchannel to improve specificity and reduce aggregation, wherein the target nucleic acid is hybridized with the oligonucleotides connected to the fixed beads. Therefore, in some embodiments, the sample nucleic acid is flowed through the microfluidic channel and under the condition that the complementary target nucleic acid (if any) is hybridized with the oligonucleotides connected to the beads, by being connected to the fixed beads of the oligonucleotides. As described above, including a sufficient amount of one or more non-sequence-specific nucleic acid binding proteins in the microfluidic channel will improve specificity and reduce aggregation. As required, the beads can be fixed to the microfluidic channel. In some embodiments, the beads are reversibly fixed so that the beads, the oligonucleotides connected, and any hybridized target nucleic acids can be released after washing, thereby allowing the presence, absence or amount of the hybridized target nucleic acid to be collected and / or detected at the second position in the microfluidic channel. In some embodiments, the beads are fixed in an electric field. For example, in some embodiments, the beads are fixed using optical tweezers. Optical tweezers technology is summarized in, for example, Bustamante et al., Nature Reviews Methods Primers, Vol. 1, Article Number: 25 (2021).

[0090] The detection of amplifiable target molecules can be performed in any manner that can be used to generate a detectable signal. Preferably, the signal can be detected optically, such as by allowing an automated scanner to detect the presence, absence or quantity of the signal in each well. The signal can increase when the target molecule is present, or in other embodiments, the signal can decrease (e.g., in the case of signal quenching in the presence of the target molecule).

[0091] In certain embodiments, the reaction mixture in which hybridization occurs includes one or more optically detectable reagents, such as fluorescent agents, phosphorescent agents, chemiluminescent agents, etc., and the reaction mixture may optionally be partitions. Many reagents (e.g., dyes, probes, or indicators) are known in the art and can be used. (See, e.g., Invitrogen, The Handbook A Guide to Fluorescent Probes and Labeling Technologies, 10th edition (2005)). Fluorescent agents may include various organic and / or inorganic small molecules or various fluorescent proteins and derivatives thereof. In certain embodiments, the reagent is a fluorophore. A large amount of fluorophores have been reported in the literature, and there are many fluorophores that can be easily obtained from commercial suppliers in the biotechnology industry. References to fluorophores include Cardullo et al., Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988); Dexter, DL, J. of Chemical Physics 21:836-850 (1953); Hochstrasser et al., Biophysical Chemistry 45:133-141 (1992); Selvin, P, Methods in Enzymology 45:134-141 (1993); Enzymology 246:300-334 (1995); Steinberg, I. Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L. Ann. Rev. Biochem., 47:819-846 (1978); Wang et al., Tetrahedron Letters 31:6493-6496 (1990); Wang et al., Anal. Chem. 67:1197-1203 (1995). Non-limiting examples of fluorophores include cyanines, fluoresceins (e.g., 5′-carboxyfluorescein (FAM), Oregon Green, and Alexa 488), HEX, rhodamines (e.g., N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC)), eosin, coumarin, pyrene, tetrapyrrole, arylmethine, oxazines, polymer dots, and quantum dots.

[0092] In some embodiments, the detectable agent is an intercalator. The intercalator generates a signal when intercalated into a double-stranded nucleic acid. Exemplary intercalators include, for example, 9-aminoacridine, ethidium bromide, phenanthridine dyes, EvaGreen, PICO GREEN (P-7581, Molecular Probes), EB (E-8751, Sigma), propidium iodide (P-4170, Sigma), acridine orange (A-6014, Sigma), thiazole orange, oxazole yellow, 7-aminoactinomycin D (A-1310, Molecular Probes), cyanine dyes (e.g., TOTO, YOYO, BOBO and POPO), SYTO, SYBR Green I (U.S. Pat. No. 5,436,134: N′,N′-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine), SYBR Green II (U.S. Pat. No. 5,658,751), SYBRDX, OliGreen, CyQuant GR, SYTOX Green, SYTO9, SYTO10, SYTO17, SYBR14, FUN-1, DEAD Red, hexidinium iodide, ethidium bromide, dihydroethidium, homodimeric ethidium, 9-amino-6-chloro-2-methoxyacridine, DAPI, DIPI, indole dyes, imidazole dyes, actinomycin D, hydroxystilbamidine, LDS 751 (U.S. Pat. No. 6,210,885), and the dyes described in: Georgiou, Photochemistry and Photobiology, 26:59-68, Pergamon Press (1977); Kubota et al., Biophys. Chem., 6:279-284 (1977); Genest et al., Nuc. Ac. Res., 13:2603-2615 (1985); Asseline, EMBO J., 3:795-800 (1984); Richardson et al., U.S. Pat. No. 4,257,774; and Letsinger et al., U.S. Pat. No. 4,547,569.

[0093] One method for detecting amplification products is the 5′-3′ exonuclease “hydrolysis” PCR assay (also known as TaqMan TMAssay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al., PNAS USA 88:7276-7280 (1991); Lee et al., Nucleic Acids Res. 21:3761-3766 (1993)). This assay uses a dual-labeled fluorescent probe (TaqMan TM The accumulation of specific PCR products is detected by hybridization and cleavage of a fluorescent probe. The fluorescent probe consists of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye. During PCR, when and only when this probe hybridizes to the segment being amplified, the probe is cleaved under the 5'-nuclease activity of the DNA polymerase. Cleavage of the probe produces an increase in the fluorescence intensity of the reporter dye.

[0094] Another method that relies on the use of energy transfer to detect amplification products is the "beacon probe" method described by Tyagi and Kramer in Nature Biotech. 14:303-309 (1996), which is also the subject of U.S. Pat. Nos. 5,119,801 and 5,312,728. This method uses an oligonucleotide hybridization probe that can form a hairpin structure. At one end (5' or 3' end) of the hybridization probe, there is a donor fluorophore, and at the other end, there is an acceptor moiety. In the case of the Tyagi and Kramer method, this acceptor moiety is a quencher, i.e., the acceptor absorbs the energy released by the donor, but then does not fluoresce itself. Thus, when the beacon is in the open conformation, the fluorescence of the donor fluorophore is detectable, while when the beacon is in the hairpin (closed) conformation, the fluorescence of the acceptor fluorophore is quenched. When used in PCR, a molecular beacon probe that hybridizes to one strand of the PCR product is in an open conformation and detects fluorescence, while a molecular beacon probe that remains unhybridized will not fluoresce (Tyagi and Kramer, Nature Biotechnology 14: 303-306 (1996)). As a result, the amount of fluorescence will increase as the amount of PCR product increases, and can therefore be used as a measure of the progress of PCR. Those skilled in the art will recognize that other methods of quantitative amplification are also available.

[0095] Thus, in some embodiments, the detectable agent is a molecular beacon oligonucleotide probe. The "beacon probe" approach relies on the use of energy transfer. This approach uses an oligonucleotide hybridization probe that can form a hairpin structure. At one end (5' or 3' end) of the hybridization probe, there is a donor fluorophore, and at the other end, there is an acceptor moiety. In the case of the Tyagi and Kramer methods, this acceptor moiety is a quencher, i.e., the acceptor absorbs the energy released by the donor, but then does not fluoresce itself. Thus, when the beacon is in an open conformation, the fluorescence of the donor fluorophore is detectable, and when the beacon is in a hairpin (closed) conformation, the fluorescence of the acceptor fluorophore is quenched.

[0096] In some embodiments, sample nucleic acid can be detected using a specific high sensitivity enzyme reporter unlocking (Specific High Sensitivity Enzymatic Reporter UnLOCKing, SHERLOCK) method. This method provides an in vitro nucleic acid detection platform with high (or single molecule) sensitivity based on nucleic acid amplification and incidental cleavage of reporter ssDNA, thereby allowing real-time detection of targets. The method of using CRISPR in SHERLOCK is described in detail in, for example, Gootenberg et al., "Nucleic acid detection with CRISPR-Cas13a / C2c2", Science, 356(6336): 438-442 (2017) and U.S. Patent Publication No. 20180340219.

[0097] In some embodiments, during amplification, one or more oligonucleotides are hybridized to a target nucleic acid in the presence of a sufficient amount of a non-sequence-specific nucleic acid binding protein, and the resulting amplicon is subsequently detected by nucleotide sequencing, such as next generation sequencing. Methods for high throughput sequencing and genotyping are known in the art. For example, such sequencing techniques include, but are not limited to, pyrosequencing, ligation sequencing, single molecule sequencing, synthetic sequencing (SBS), massively parallel cloning, massively parallel single molecule SBS, massively parallel single molecule real-time, massively parallel single molecule real-time nanopore technology, and the like. Morozova and Marra provide a review of some such techniques in Genomics, 92: 255 (2008), which is incorporated herein by reference in its entirety.

[0098] Exemplary DNA sequencing techniques include fluorescence-based sequencing methods (see, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor Laboratory Press, New York; the document is incorporated herein by reference in its entirety). In some embodiments, automated sequencing techniques understood in the art are utilized. In some embodiments, the present technology provides parallel sequencing of partitioned amplicons (PCT Publication No. WO 2006 / 0841,32, the document is incorporated herein by reference in its entirety). In some embodiments, DNA sequencing is achieved by parallel oligonucleotide extension (see, e.g., U.S. Pat. Nos. 5,750,341; and 6,306,597, both of which are incorporated herein by reference in their entirety). Additional examples of sequencing techniques include: Church polony technology (Mitra et al., 2003, Analytical Biochemistry). Biochemistry, 320, 55-65; Shendure et al., 2005 Science, 309, 1728-1732; and U.S. Pat. Nos. 6,432,360; 6,485,944; and 6,511,803; the entireties of which are incorporated herein by reference); 454 pico-titer pyrosequencing (Margulies et al., 2005 Nature, 437, 376-380; U.S. Publication No. 2005 / 0130173; the entireties of which are incorporated herein by reference); S olexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Pat. Nos. 6,787,308; and 6,833,246; the entireties of which are incorporated herein by reference); Lynx massively parallel tag sequencing technology (Brenner et al. (2000). Nature Biotechnology 18: 630-634; U.S. Pat. Nos. 5,695,934; and 5,714,330; the entireties of which are incorporated herein by reference); and Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acids Research 28, E87; WO 2000 / 018957; the entireties of which are incorporated herein by reference).

[0099] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes therefrom will occur to those skilled in the art and are included within the spirit and scope of the present application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method for introducing oligonucleotide-ligated beads into partitions, the method comprising: providing a bulk solution comprising (i) a plurality of beads of said linked oligonucleotides and (ii) a non-sequence-specific nucleic acid binding protein in an amount sufficient to reduce aggregation of said beads; as well as The beads are introduced into the partitions such that the number of beads in the partitions is less than 5 beads per partition on average. The method according to claim 1 , wherein the partition is a microwell or a droplet.

3. The method of claim 1, wherein at least most of the oligonucleotides coated on the beads have the same sequence, and oligonucleotides on different beads can be distinguished by barcode sequences on the oligonucleotides.

4. The method according to any one of claims 1 to 3, wherein the oligonucleotide comprises a free 3' end comprising at least 6 consecutive thymine nucleotides forming a polythymidylate sequence.

5. The method according to any one of claims 1 to 4, wherein during said introducing, said partitions comprise a single cell per partition. The method according to claim 5 , wherein after said introducing, cells in said partitions are lysed and said oligonucleotides in said partitions are hybridized with target nucleic acids from said cells.

7. The method of claim 5, further comprising adding an additional amount of the non-sequence-specific nucleic acid binding protein to the partitions prior to the hybridization. The method of claim 6 , wherein the oligonucleotide is attached to the bead prior to the hybridization.

9. The method of claim 6, wherein the oligonucleotide is released from the bead prior to the hybridization.

10. The method of claim 6, further comprising extending the oligonucleotide in a template-specific manner using a polymerase to connect the reverse complementary sequence of the target nucleic acid to the oligonucleotide.

11. The method of claim 6, wherein the nucleic acid is RNA.

12. The method of claim 6, wherein the nucleic acid is DNA.

13. The method according to any one of claims 1 to 12, wherein the non-sequence specific nucleic acid binding protein is selected from the group consisting of: RecA, RecO, RecN, RadA, Rad51, Rad52 and UvsX.

14. A solution comprising (i) a plurality of beads to which oligonucleotides are attached and (ii) a non-sequence-specific nucleic acid binding protein in an amount sufficient to reduce aggregation of the beads.

15. The solution of claim 14, wherein the oligonucleotide comprises a free 3' end, the free 3' end comprising at least 6 consecutive thymine nucleotides, the thymine nucleotides forming a poly-thymidylate sequence.

16. The solution of claim 14, wherein the non-sequence specific nucleic acid binding protein is selected from the group consisting of RecA, RecO, RecN, RadA, Rad51, Rad52 and UvsX.

17. A method of introducing oligonucleotide-ligated beads into partitions, the method comprising: providing a plurality of partitions, wherein the partitions include (i) beads to which oligonucleotides are attached and (ii) a non-sequence-specific nucleic acid binding protein, and (iii) a target nucleic acid; as well as In the presence of the non-sequence-specific nucleic acid binding protein, the oligonucleotide is hybridized to the target nucleic acid.

18. The method of claim 17, wherein the number of beads in the partitions is less than 5 beads per partition on average.

19. The method of claim 17, wherein the method comprises releasing the oligonucleotide from the bead prior to the hybridization.

20. The method of claim 17, wherein the oligonucleotide comprises a free 3' end, the free 3' end comprising at least 6 consecutive thymine nucleotides, the thymine nucleotides forming a poly-thymidylate sequence.

21. The method of claim 17, wherein at least a majority of the oligonucleotides attached to the beads have the same sequence, and oligonucleotides on different beads can be distinguished by barcode sequences on the oligonucleotides.

22. The method of claim 17, wherein the partitions are microwells or droplets.

23. The method according to any one of claims 17 to 22, wherein the non-sequence specific nucleic acid binding protein is selected from the group consisting of: RecA, RecO, RecN, RadA, Rad51, Rad52 and UvsX.

24. A reaction mixture comprising (i) beads to which oligonucleotides are attached and (ii) a non-sequence-specific nucleic acid binding protein, and (iii) a target nucleic acid.

25. The reaction mixture of claim 24, wherein the non-sequence specific nucleic acid binding protein is selected from the group consisting of RecA, RecO, RecN, RadA, Rad51, Rad52 and UvsX.

26. A plurality of partitions, wherein the partitions contain a reaction mixture according to claim 24 or 25, wherein at least a majority of the oligonucleotides attached to the beads in the partitions have the same sequence, and the oligonucleotides on different beads in different partitions can be distinguished by a barcode sequence on the oligonucleotides.

27. The plurality of partitions of claim 26, wherein the partitions are microwells or droplets.

28. A method for detecting nucleic acid in a microfluidic channel, the method comprising: flowing a nucleic acid sample in a microfluidic channel past beads of immobilized linked oligonucleotides in the presence of a non-sequence-specific nucleic acid binding protein under conditions sufficient to allow the oligonucleotides to specifically hybridize to target nucleic acids in the nucleic acid sample; as well as Hybridization of the target nucleic acid to the oligonucleotide is detected.

29. The method of claim 28, wherein the beads of linked oligonucleotides are immobilized in the microfluidic channel by optical tweezers.

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