Methods for characterizing cell-free nucleic acid fragments
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.
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.
2. 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.
3. 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.
4. The method of claim 2 , wherein said amplification is performed by polymerase chain reaction (PCR).
5. The method of claim 1 , wherein said first oligonucleotide bait or said second oligonucleotide bait is conjugated to an affinity tag.
6. The method of claim 5 , wherein said affinity tag is biotin.
7. The method of claim 1 , wherein said first oligonucleotide bait and said second oligonucleotide bait are conjugated to a solid surface.
8. The method of claim 7 , wherein said solid surface is a bead.
9. The method of claim 7 , wherein said solid surface is a planar surface.
10. 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.
11. The method of claim 1 , further comprising obtaining said cfDNA fragments from plasma.
12. The method of claim 1 , wherein said genomic region comprises at least part of a promotor or a transcriptional start site.
13. The method of claim 1 , wherein said cfDNA fragments are from a cell that has undergone necrosis or apoptosis.
14. 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.
15. 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).
16. 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.
17. The method of claim 1 , wherein said first oligonucleotide bait or said second oligonucleotide bait are conjugated to a fluorescent label.
18. The method of claim 10 , wherein said genomic region is associated with a pathological condition.
19. The method of claim 1 , further comprising comparing said amount of said cfDNA fragments with a reference database.