# US 11,046,999 B2 · Methods and compositions for genomic target enrichment and selective DNA sequencing > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,046,999 B2 - Document ID: US11046999B2 - Status: Granted - Kind: B2 - Application number: 15/268,403 - Filed: 2016-09-16 - Published: 2017-03-16 - Granted: 2021-06-29 - Earliest claimed: 2015-09-16 - Assignees: PetaOmics, Inc. - Applicants: PetaOmics, Inc. - Inventors: Paul M. Lizardi; Brent W. Ferguson - Web page: https://patentgrep.com/patent/US11046999B2 ## CPC - C12Q 1/6839: Triple helix formation or other higher order conformations in hybridisation assays - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract It has been established that one or more large double stranded DNA fragments (each 2,000 to 40,000 base pairs in size) can be captured and isolated from genomic DNA fragments using sequence specific PNA hybridization probes. Compositions and methods for enrichment of a multiplicity of long DNA sequences selected from the genome of any eukaryote are provided. Capture is performed using multiple PNA molecules with gamma-modified chiral backbones, comprising a mixture of neutral and positive chemical groups. Two or more PNA probes with covalently bound haptens, preferably biotin, target each DNA domain of interest for capture, isolation, and subsequent sequencing analysis of the multiplicity of enriched targets, including DNA methylation sequencing. The methods include enhancement of probe-DNA binding specificity through single strand binding proteins (SSB). ## Claims ### Claim 1 A peptide nucleic acid (PNA) hybridization probe comprising at or between 10 to 26 peptide nucleic acid residues, wherein the PNA probe is designed to target a sequence in a nucleic acid fragment, wherein the PNA probe comprises two or more peptide nucleic acid residues that are derivatized with a charged moiety on, independently for each of the peptide nucleic acid residues derivatized with a charged moiety, the alpha, beta, or gamma carbon, and one or more peptide nucleic acid residues that are derivatized with a neutral moiety on, independently for each of the peptide nucleic acid residues derivatized with a neutral moiety, the alpha, beta, or gamma carbon, wherein there is an average of at or between 1.8 to 4.0 peptide nucleic acid residues that are not derivatized with a charged moiety between every peptide nucleic acid residue that is derivatized with a charged moiety, and wherein the PNA probe comprises one or more capture tags. ### Claim 2 (depends on 1) The PNA probe of claim 1, wherein the probe comprises at or between 16 to 22 peptide nucleic acid residues. ### Claim 3 (depends on 1) The PNA probe of claim 1, wherein the probe comprises 18 or 19 peptide nucleic acid residues. ### Claim 4 (depends on 1) The PNA probe of claim 1, wherein three, four, five, or six of the peptide nucleic acid residues are derivatized with the charged moieties, wherein one or more of the peptide nucleic acid residues derivatized with the charged moieties are selected from the group consisting of gamma-L-lysine PNA, gamma-L-thialysine PNA, and combinations thereof, wherein at or between two to six of the peptide nucleic acid residues that are not derivatized with the charged moieties are derivatized with diethylene glycol, and wherein the capture tag is biotin. ### Claim 5 (depends on 1) The PNA probe of claim 1, wherein four of the peptide nucleic acid residues are gamma-L-lysine PNA, wherein four of the peptide nucleic acid residues are derivatized with diethylene glycol, and wherein the capture tag is biotin. ### Claim 6 (depends on 1) The PNA probe of claim 1, wherein four of the peptide nucleic acid residues are gamma-L-thialysine PNA, wherein four of the peptide nucleic acid residues are derivatized with diethylene glycol, and wherein the capture tag is biotin. ### Claim 7 (depends on 1) The PNA probe of claim 1, wherein there is an average of at or between 0.4 to 1.5 peptide nucleic acid residues that are not derivatized with a moiety between every peptide nucleic acid residue that is derivatized with a moiety. ### Claim 8 (depends on 1) The PNA probe of claim 1, wherein every peptide nucleic acid residue is derivatized with a charged moiety or a neutral moiety. ### Claim 9 (depends on 1) The PNA probe of claim 1, wherein at or between 15% to 28% of the peptide nucleic acid residues of the PNA probe are derivatized with a charged moiety. ### Claim 10 (depends on 1) The PNA probe of claim 1, wherein there are at least two peptide nucleic acid residues that are not derivatized with a charged moiety between every peptide nucleic acid residue that is derivatized with a charged moiety. ### Claim 11 (depends on 1) The PNA probe of claim 1, wherein one or more of the peptide nucleic acid residues that are derivatized with the charged moiety are derivatized with the charged moiety on the gamma carbon. ### Claim 12 (depends on 1) The PNA probe of claim 1, wherein one or more of the peptide nucleic acid residues that are derivatized with the charged moieties are L-thialysine peptide nucleic acid residues. ### Claim 13 (depends on 1) The PNA probe of claim 1, wherein one or more of the peptide nucleic acid residues that are derivatized with the charged moieties are L-lysine peptide nucleic acid residues. ### Claim 14 (depends on 1) The PNA probe of claim 1, wherein at or between 4% to 85% of the peptide nucleic acid residues of the PNA probe are derivatized with a neutral moiety. ### Claim 15 (depends on 1) The PNA probe of claim 1, wherein one or more of the peptide nucleic acid residues that are derivatized with a neutral moiety are derivatized on the gamma carbon. ### Claim 16 (depends on 1) The PNA probe of claim 1, wherein one or more of the neutral moieties is a short-chain oligoethylene moiety. ### Claim 17 (depends on 16) The PNA probe of claim 16, wherein one or more of the short-chain oligoethylene moieties are diethylene glycol. ### Claim 18 (depends on 1) The PNA probe of claim 1, wherein the capture tag is biotin or streptavidin. ### Claim 19 (depends on 1) The PNA probe of claim 1, wherein the PNA probe targets (a) a sequence in human genomic DNA located in the MHC region of chromosome 6; (b) a sequence in human genomic DNA associated with one or more diseases or conditions or having a known correlation with development of one or more disease or conditions, wherein the diseases or conditions are selected from the group consisting of autoimmune diseases, diabetes, metabolic syndrome, and cancer; (c) a sequence in human genomic DNA at different positions that map to a multiplicity of enhancer elements associated with disease risk for autoimmune diseases; (d) a sequence in human genomic DNA at different positions that map to a multiplicity of enhancer elements associated with disease risk for diabetes and the metabolic syndrome; (e) a sequence in human genomic DNA at different positions that map to a multiplicity of enhancer elements associated with the differentiation of different subsets of white blood cells; (f) a sequence in human mitochondrial DNA; (g) a sequence in dog mitochondrial DNA; or (h) a sequence in genomic DNA of one or more parasites selected from the group consisting of bacteria, archaea, fungi, protozoa, or mixtures thereof. ### Claim 20 (depends on 1) The PNA probe of claim 1, wherein the PNA probe targets a sequence in genomic DNA of one or more parasites selected from the group consisting of bacteria, archaea, fungi, protozoa, or mixtures thereof, wherein the bacteria is one or more species of bacteria present in human oral cavity, human airway, human urogenital tract, human blood, or human feces. ### Claim 21 (depends on 1) A set of two or more PNA probes, wherein at least one of the PNA probes is a PNA probe of claim 1, wherein the PNA probes in the same set of two or more PNA probes are designed to target a different sequence in the same nucleic acid fragment, wherein the PNA probes in different sets of two or more PNA probes are designed to target different nucleic acid fragments. ### Claim 22 (depends on 21) The set of claim 21, wherein all of the PNA probes are a PNA probe, wherein each PNA probe independently (a) comprises at or between 10 to 26 peptide nucleic acid residues, (b) is designed to target a sequence in a nucleic acid fragment, (c) comprises one or more peptide nucleic acid residues that are derivatized with a charged moiety on the alpha, beta, or gamma carbon or combinations thereof, and one or more peptide nucleic acid residues that are derivatized with a neutral moiety on the alpha, beta, or gamma carbon, or combinations thereof, and (d) comprises one or more capture tags. ### Claim 23 (depends on 21) The set of claim 21, wherein in one or more of the PNA probes there is an average of at or between 1.0 to 5.0 peptide nucleic acid residues that are not derivatized with a charged moiety between every peptide nucleic acid residue that is derivatized with a charged moiety. ### Claim 24 (depends on 21) The set of claim 21, wherein in one or more of the PNA probes there is an average of at or between 0.5 to 1.5 peptide nucleic acid residues that are not derivatized with a moiety between every peptide nucleic acid residue that is derivatized with a moiety. ### Claim 25 (depends on 21) The set of claim 21, wherein independently in one or more of the PNA probes one or more of the peptide nucleic acid residues that are derivatized with the charged moiety are derivatized with the charged moiety on the gamma carbon. ### Claim 26 (depends on 21) The set of claim 21, wherein in all of the PNA probes one or more of the peptide nucleic acid residues that are derivatized with the charged moiety are derivatized with the charged moiety on the gamma carbon. ### Claim 27 (depends on 21) The set of claim 21, wherein in one or more of the PNA probes one or more of the peptide nucleic acid residues that are derivatized with the charged moieties are L-thialysine peptide nucleic acid residues. ### Claim 28 (depends on 21) The set of claim 21, wherein in one or more of the PNA probes one or more of the peptide nucleic acid residues that are derivatized with the charged moieties are L-lysine peptide nucleic acid residues. ### Claim 29 (depends on 21) The set of claim 21, wherein in all of the PNA probes one or more of the peptide nucleic acid residues that are derivatized with the charged moieties are L-thialysine peptide nucleic acid residues. ### Claim 30 (depends on 21) The set of claim 21, wherein in all of the PNA probes one or more of the peptide nucleic acid residues that are derivatized with the charged moieties are L-lysine peptide nucleic acid residues. ### Claim 31 (depends on 21) The set of claim 21, wherein independently in one or more of the PNA probes one or more of the peptide nucleic acid residues that are derivatized with the short-chain oligoethylene moiety are derivatized with the short-chain oligoethylene moiety on the gamma carbon. ### Claim 32 (depends on 21) The set of claim 21, wherein in all of the PNA probes one or more of the peptide nucleic acid residues that are derivatized with the short-chain oligoethylene moiety are derivatized with the short-chain oligoethylene moiety on the gamma carbon. ### Claim 33 (depends on 21) The set of claim 21, wherein in one or more of the PNA probes one or more of the short-chain oligoethylene moieties are diethylene glycol. ### Claim 34 (depends on 21) The set of claim 21, wherein in all of the PNA probes one or more of the short-chain oligoethylene moieties are diethylene glycol. ### Claim 35 (depends on 21) The set of claim 21, wherein the capture tag is biotin or streptavidin. ### Claim 36 (depends on 21) The set of claim 21, wherein the PNA probes target (a) sequences in human genomic DNA located in the MHC region of chromosome 6; (b) sequences in human genomic DNA associated with one or more diseases or conditions or having a known correlation with development of one or more disease or conditions, wherein the diseases or conditions are selected from the group consisting of autoimmune diseases, diabetes, metabolic syndrome, and cancer; (c) sequences in human genomic DNA at different positions that map to a multiplicity of enhancer elements associated with disease risk for autoimmune diseases; (d) sequences in human genomic DNA at different positions that map to a multiplicity of enhancer elements associated with disease risk for diabetes and the metabolic syndrome; (e) sequences in human genomic DNA at different positions that map to a multiplicity of enhancer elements associated with the differentiation of different subsets of white blood cells; (f) sequences in human mitochondrial DNA; (g) sequences in dog mitochondrial DNA; or (h) sequences in genomic DNA of one or more parasites selected from the group consisting of bacteria, archaea, fungi, protozoa, or mixtures thereof. ### Claim 37 (depends on 21) The set of claim 21, wherein the PNA probes target sequences in genomic DNA of one or more parasites selected from the group consisting of bacteria, archaea, fungi, protozoa, or mixtures thereof, wherein the bacteria is one or more species of bacteria present in human oral cavity, human airway, human urogenital tract, human blood, or human feces. ### Claim 38 (depends on 21) A method of selectively enriching one or more nucleic acid fragments from a mixture of nucleic acid fragments, the method comprising: (a) bringing into contact one or more sets of two or more PNA probes of claim 21 with a first nucleic acid sample to form a reaction mix; (b) incubating the reaction mix under conditions that allow target-specific strand invasion binding by the PNA probes to their target sequence in a nucleic acid fragment, thereby forming nucleic acid fragments bound by invading PNA probes; (c) capturing the nucleic acid fragments bound by PNA probes via the capture tag and removing the uncaptured components of the reaction mix from the captured nucleic acid fragments bound by PNA probes; (d) eluting the captured nucleic acid fragments from the PNA probes to form an enriched nucleic acid sample, wherein nucleic acid fragments targeted by the PNA probes are enriched in the enriched nucleic acid sample as compared to the first nucleic acid sample. ### Claim 39 (depends on 38) The method of claim 38 wherein the reaction mix further comprises a single-strand binding protein. ### Claim 40 (depends on 38) The method of claim 38, wherein the first nucleic acid sample has high sequence complexity. ### Claim 41 (depends on 38) The method of claim 38, wherein the first nucleic acid sample includes double stranded DNA. ### Claim 42 (depends on 41) The method of claim 41, wherein the double stranded DNA has never been completely denatured or never been substantially denatured. ### Claim 43 (depends on 38) The method of claim 38, wherein the first nucleic acid sample includes genomic DNA. ### Claim 44 (depends on 38) The method of claim 38, wherein the enriched nucleic acid fragments have an average length of at least 2,000 base pairs. ### Claim 45 (depends on 38) The method of claim 38, wherein the enriched nucleic acid fragments have an average length of at least 10,000 base pairs. ### Claim 46 (depends on 38) The method of claim 38, wherein the enriched nucleic acid fragments have an average length of at least 15,000 base pairs. ### Claim 47 (depends on 38) The method of claim 38, wherein each of the enriched nucleic acid fragments has a length of at least 2,000 base pairs. ### Claim 48 (depends on 38) The method of claim 38, wherein the enriched nucleic acid sample comprises a molar ratio of targeted to non-targeted nucleic acid fragments that is between 50:1 and 150:1. ### Claim 49 (depends on 38) The method of claim 38 further comprising, following step (b) and prior to step (c), removing unbound PNA probes from the reaction mix. ### Claim 50 (depends on 38) The method of claim 38 further comprising, simultaneous with capturing the nucleic acid fragments bound by PNA probes, capturing unbound PNA probes via the capture tag. ### Claim 51 (depends on 38) The method of claim 38, wherein eluting the bound nucleic acid fragments in step (d) is carried out using Herculase II DNA polymerase. ### Claim 52 (depends on 38) The method of claim 38, wherein eluting the bound nucleic acid fragments in step (d) is carried out by de-protonation of the charged moiety by raising the pH. ### Claim 53 (depends on 38) The method of claim 38 further comprising amplifying one or more of the nucleic acid fragments in the enriched nucleic acid sample. ### Claim 54 (depends on 53) The method of claim 53, wherein substantially all of the nucleic acid fragments in the enriched nucleic acid sample are amplified. ### Claim 55 (depends on 53) The method of claim 53, wherein the nucleic acid fragments are amplified by whole genome amplification. ### Claim 56 (depends on 38) The method of claim 38, wherein the nucleic acid sample comprises ILLUMINA-MOLECULO® adapter-ligated nucleic acid fragments. ### Claim 57 (depends on 38) The method of claim 38, wherein the nucleic acid sample comprises nucleic acid fragments that have been end-repaired and purified according to one or more protocols for PACIFIC BIOSCIENCES® Library Preparation. ### Claim 58 (depends on 38) The method of claim 38, wherein the nucleic acid sample comprises PACBIO® hairpin adapter-ligated nucleic acid fragments. ### Claim 59 (depends on 38) The method of claim 38, further comprising, following step (c) and prior to step (d), ligating PACBIO® hairpin adapters to the captured nucleic acid. ### Claim 60 (depends on 21) A kit comprising (a) the set of two or more PNA probes of claim 21; and (b) instructions for performing a method, wherein the method comprises (i) bringing into contact the set with a first nucleic acid sample to form a reaction mix; (ii) incubating the reaction mix under conditions that allow target-specific strand invasion binding by the PNA probes to their target sequence in a nucleic acid fragment, thereby forming nucleic acid fragments bound by invading PNA probes; (iii) capturing the nucleic acid fragments bound by PNA probes via the capture tag and removing the uncaptured components of the reaction mix from the captured nucleic acid fragments bound by PNA probes; (iv) eluting the captured nucleic acid fragments from the PNA probes to form an enriched nucleic acid sample, wherein nucleic acid fragments targeted by the PNA probes are enriched in the enriched nucleic acid sample as compared to the first nucleic acid sample. ### Claim 61 (depends on 60) The kit of claim 60 further comprising one of more enzymes or proteins for performing one or more steps in the method. ### Claim 62 A method of selectively enriching one or more nucleic acid fragments from a mixture of nucleic acid fragments, the method comprising: (a) bringing into contact one or more sets of two or more peptide nucleic acid (PNA) hybridization probes with a first nucleic acid sample to form a reaction mix, wherein the PNA probes in the same set of two or more PNA probes are designed to target a different sequence in the same nucleic acid fragment, wherein the PNA probes in different sets of two or more PNA probes are designed to target different nucleic acid fragments, wherein the PNA probes each comprise one or more capture tags, wherein at least one of the PNA probes includes two or more peptide nucleic acid residues that are derivatized with a charged moiety on the alpha carbon, beta carbon, gamma carbon, or combinations thereof and one or more peptide nucleic acid residues that are derivatized with a neutral moiety on the alpha carbon, beta carbon, gamma carbon, or combinations thereof, and wherein there is an average of at or between 1.8 to 4.0 peptide nucleic acid residues that are not derivatized with a charged moiety between every peptide nucleic acid residue that is derivatized with a charged moiety; (b) incubating the reaction mix under conditions that allow target-specific strand invasion binding by the PNA probes to their target sequence in a nucleic acid fragment, thereby forming nucleic acid fragments bound by invading PNA probes; (c) capturing the nucleic acid fragments bound by PNA probes via the capture tag and removing the uncaptured components of the reaction mix from the captured nucleic acid fragments bound by PNA probes; (d) eluting the captured nucleic acid fragments from the PNA probes to form an enriched nucleic acid sample, wherein nucleic acid fragments targeted by the PNA probes are enriched in the enriched nucleic acid sample as compared to the first nucleic acid sample. ### Claim 63 (depends on 62) The method of claim 62, wherein the PNA probes in at least one of the sets of two or more PNA probes have 18 or 19 peptide nucleic acid residues, wherein at or between three to five of the peptide nucleic acid residues of the PNA probes in the at least one of the sets of two or more PNA probes are derivatized with the charged moieties, wherein one or more of the peptide nucleic acid residues derivatized with charged moieties are selected from the group consisting of gamma-L-lysine PNA, gamma-L-thialysine PNA, and combinations thereof, wherein at or between two to six of the peptide nucleic acid residues of the PNA probes in the at least one of the sets of two or more PNA probes that are not derivatized with the charged moieties are derivatized with diethylene glycol, and wherein the capture tag of the PNA probes in at least one of the sets of two or more PNA probes is biotin. ### Claim 64 (depends on 1) The PNA probe of claim 1, wherein the probe comprises one or more peptide nucleic acid residues that are derivatized with a negatively charged moiety on the alpha, beta, or gamma carbon or combinations thereof. ### Claim 65 (depends on 64) The PNA probe of claim 64, wherein the negatively charged moiety is glutamic acid or derivatives and variants thereof. ### Claim 66 (depends on 1) The PNA probe of claim 1, wherein one or more of the peptide nucleic acid residues that are derivatized with the charged moieties are glutamic acid peptide nucleic acid residues. ### Claim 67 (depends on 1) The PNA probe of claim 1, wherein the probe comprises 18, 19, or 20 peptide nucleic acid residues. ### Claim 68 (depends on 67) The PNA probe of claim 67, wherein five or six of the peptide nucleic acid residues are derivatized with the charged moieties. ### Claim 69 (depends on 68) The PNA probe of claim 68, wherein one or more of the peptide nucleic acid residues derivatized with the charged moieties are selected from the group consisting of gamma-L-lysine PNA, gamma-L-thialysine PNA, and combinations thereof. ### Claim 70 (depends on 69) The PNA probe of claim 69, wherein one or more of the peptide nucleic acid residues that are derivatized with the charged moieties are glutamic acid peptide nucleic acid residues. ### Claim 71 (depends on 70) The PNA probe of claim 70, wherein at or between two to six of the peptide nucleic acid residues that are not derivatized with the charged moieties are derivatized with diethylene glycol, and wherein the capture tag is biotin. ### Claim 72 (depends on 1) The PNA probe of claim 1, wherein the probe comprises at or between 15 to 25 peptide nucleic acid residues. ### Claim 73 (depends on 1) The PNA probe of claim 1, wherein there is an average of at or between 0.8 to 2.0 peptide nucleic acid residues that are not derivatized with a moiety between every peptide nucleic acid residue that is derivatized with a moiety. ### Claim 74 (depends on 1) The PNA probe of claim 1, wherein at or between 31% to 50% of the peptide nucleic acid residues of the PNA probe are derivatized with a charged moiety or a neutral moiety. ### Claim 75 (depends on 1) The PNA probe of claim 1, wherein at or between 15% to 32% of the peptide nucleic acid residues of the PNA probe are derivatized with a charged moiety. ### Claim 76 (depends on 21) The set of claim 21, wherein all of the PNA probes are a PNA probe, wherein each PNA probe independently (a) comprises at or between 15 to 25 peptide nucleic acid residues, (b) is designed to target a sequence in a nucleic acid fragment, (c) comprises one or more peptide nucleic acid residues that are derivatized with a charged moiety on the alpha, beta, or gamma carbon or combinations thereof, and one or more peptide nucleic acid residues that are derivatized with a neutral moiety on the alpha, beta, or gamma carbon, or combinations thereof, and (d) comprises one or more capture tags. ### Claim 77 (depends on 21) The set of claim 21, wherein in one or more of the PNA probes there is an average of at or between 0.8 to 2.0 peptide nucleic acid residues that are not derivatized with a moiety between every peptide nucleic acid residue that is derivatized with a moiety. ### Claim 78 (depends on 21) The set of claim 21, wherein in one or more of the PNA probes at or between 31% to 50% of the peptide nucleic acid residues of the PNA probe are derivatized with a charged moiety or a neutral moiety. ### Claim 79 (depends on 1) The PNA probe of claim 1, wherein three, four, five, or six of the peptide nucleic acid residues are derivatized with the charged moieties on, independently for each of the peptide nucleic acid residues derivatized with a charged moiety, the alpha, beta, or gamma carbon.