# US 11,993,818 B2 · Methods for characterizing cell-free nucleic acid fragments > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,993,818 B2 - Document ID: US11993818B2 - Status: Granted - Kind: B2 - Application number: 18/336,901 - Filed: 2023-06-16 - Published: 2023-11-30 - Granted: 2024-05-28 - Earliest claimed: 2020-05-22 - Assignees: Aqtual, Inc. - Applicants: Aqtual, Inc. - Inventors: Diana Abdueva - Web page: https://patentgrep.com/patent/US11993818B2 ## CPC - C12Q 1/6883: for diseases caused by alterations of genetic material - G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations - C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence) - C12Q 1/6869: Methods for sequencing - G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids - C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism - C12Q 2600/158: Expression markers - 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 - G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY ## Abstract The present disclosure provides methods and systems for various uses of cell-free nucleic acid (cfNA). Functional typing of cfNA fragmentation patterns may be utilized in the non-invasive detection, diagnosis, and monitoring of disease. One embodiment may determine a stage of cancer in a subject, the progression of cancer in a subject, or the responsiveness to treatment of a cancer in a subject. Another embodiment disclosed herein may include sequencing-free diagnostic methods. ## Claims ### Claim 1 A method of characterizing cell-free DNA (cfDNA) fragments comprising a sequence of a genomic region, comprising: (a) contacting said cfDNA fragments with: (i) a first oligonucleotide bait that hybridizes to a first portion of said genomic region, and (ii) a second oligonucleotide bait that hybridizes to a second portion of said genomic region; and (b) detecting: (i) an amount of said cfDNA fragments that comprise said first portion of said genomic region; and (ii) an amount of said cfDNA fragments that comprise said first portion of said genomic region and said second portion of said genomic region using said first oligonucleotide bait and said second oligonucleotide bait, wherein said first nucleotide bait is configured to hybridize to a cfDNA of less than 200 nucleotides in length and a cfDNA of greater than 200 nucleotides in length, wherein said second oligonucleotide bait is configured to hybridize to said cfDNA of greater than 200 nucleotides in length preferentially over said cfDNA of less than 200 nucleotides in length, and wherein said genomic region comprises at least part of a promotor, a transcriptional start site, a DNAse I-hypersensitive site, a Pol II pausing site, a first exon, or an intron to exon boundary. ### Claim 2 (depends on 1) The method of claim 1, wherein (b) comprises amplification of said first portion of said genomic region or said second portion of said genomic region. ### Claim 3 (depends on 2) The method of claim 2, wherein said amplification is performed by loop mediated isothermal amplification, nucleic acid sequence-based amplification, strand displacement amplification, or multiple displacement amplification. ### Claim 4 (depends on 2) The method of claim 2, wherein said amplification is performed by polymerase chain reaction (PCR). ### Claim 5 (depends on 1) The method of claim 1, wherein said first oligonucleotide bait or said second oligonucleotide bait is conjugated to an affinity tag. ### Claim 6 (depends on 5) The method of claim 5, wherein said affinity tag is biotin. ### Claim 7 (depends on 1) The method of claim 1, wherein said first oligonucleotide bait and said second oligonucleotide bait are conjugated to a solid surface. ### Claim 8 (depends on 7) The method of claim 7, wherein said solid surface is a bead. ### Claim 9 (depends on 7) The method of claim 7, wherein said solid surface is a planar surface. ### Claim 10 (depends on 1) The method of claim 1, further comprising obtaining said cfDNA fragments from serum, saliva, urine, blood components, cerebrospinal fluid, pleural fluid, amniotic fluid, peritoneal fluid, ascitic fluid, abdominopelvic washings/lavage, serous effusions, or tracheobronchial or bronchoalveolar lavage. ### Claim 11 (depends on 1) The method of claim 1, further comprising obtaining said cfDNA fragments from plasma. ### Claim 12 (depends on 1) The method of claim 1, wherein said genomic region comprises at least part of a promotor or a transcriptional start site. ### Claim 13 (depends on 1) The method of claim 1, wherein said cfDNA fragments are from a cell that has undergone necrosis or apoptosis. ### Claim 14 (depends on 1) The method of claim 1, further comprising detecting an amount of said cfDNA that hybridizes with said first oligonucleotide bait or an amount of said cfDNA that hybridizes with said second oligonucleotide bait by sequencing or fluorimetry. ### Claim 15 (depends on 1) The method of claim 1, further comprising detecting an amount of said cfDNA that hybridizes with said first oligonucleotide bait or an amount of said cfDNA that hybridizes with said second oligonucleotide bait by quantitative polyermase chain reaction (qPCR) or reverse transcriptase polyermase chain reaction (rtPCR). ### Claim 16 (depends on 1) The method of claim 1, further comprising detecting an amount of said cfDNA that hybridizes with said first oligonucleotide bait or an amount of said cfDNA that hybridizes with said second oligonucleotide bait by electrophoresis. ### Claim 17 (depends on 1) The method of claim 1, wherein said first oligonucleotide bait or said second oligonucleotide bait are conjugated to a fluorescent label. ### Claim 18 (depends on 10) The method of claim 10, wherein said genomic region is associated with a pathological condition. ### Claim 19 (depends on 1) The method of claim 1, further comprising comparing said amount of said cfDNA fragments with a reference database.