Sample preparation methods, systems and compositions
Inventors
Blauwkamp, Timothy A • Sit, Rene • Vilfan, Igor D.
Assignees
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Publication Number
US-11834711-B2
Publication Date
2023-12-05
Expiration Date
Abstract
The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.
Core Innovation
The invention provides methods for detection by concurrent processing of different nucleic acid forms in a sample. A sample comprises a first nucleic acid form and a second nucleic acid form, which are denatured and then ligated with adapters to enable downstream detection. The adapters comprise different identifying sequences so that the detected nucleic acid forms are distinguished after processing.
The method performs ligating a first adapter to one end of the first nucleic acid form using a first ligase that has a preference for the first nucleic acid form, and ligating a second adapter to one end of the second nucleic acid form using a second ligase that has a preference for the second nucleic acid form. After ligation, the ligated nucleic acid forms are detected.
The approach is described in the context of concurrent analysis of DNA and RNA forms and can include distinguishing structural nucleic acid forms, including particle-protected and exosomal nucleic acids, as part of infection assessment. The disclosed nucleic-acid detection applications include cancer diagnosis and therapy tailoring, fetal health assessment, and infection assessment.
Claims Coverage
The claim set describes processing different nucleic acid forms concurrently using form-preferred ligases, different identifying adapter sequences, and detection of the ligated products. Independent and dependent claims add concurrency and reaction configuration, adapter-ligation scope, nucleic-acid-type pairings, ligase options, and sample source narrowing.
Concurrent adapter ligation of different nucleic acid forms with form-preferred ligases
A method for concurrent processing of a sample comprising a first nucleic acid form and a second nucleic acid form, including denaturing both forms; ligating a first adapter to one end of the first nucleic acid form using a first ligase that has a preference for the first nucleic acid form; ligating a second adapter to one end of the second nucleic acid form using a second ligase that has a preference for the second nucleic acid form; wherein each adapter comprises a different identifying sequence; and detecting the ligated nucleic acid forms.
Concurrent adapter ligation
The method where ligating the first adapter and ligating the second adapter occur concurrently.
Adapter ligation within a single reaction mixture
The method where ligating the first adapter and ligating the second adapter are performed within a single reaction mixture.
DNA and RNA form-paired ligase preferences
The method configured such that the first nucleic acid form is DNA and the second nucleic acid form is RNA, with DNA-preferred and RNA-preferred ligases being a DNA ligase and an RNA ligase, respectively.
Selected DNA ligase options for DNA-preferred ligation
The method where the DNA ligase is selected from PBCV-1 DNA ligase/Chlorella virus DNA ligase, CircLigase ssDNA ligase, thermostable DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, E. coli DNA ligase, or combinations thereof.
Cell-free microbial DNA as the DNA source
The method where the cell-free DNA is cell-free microbial DNA.
The core claim requires concurrent denaturation, form-preferred adapter ligation with different identifying sequences, and detection of the ligated nucleic acid forms. Dependent claims add constraints including concurrency and single-mixture operation, specify DNA-versus-RNA form pairings with corresponding preferred ligases, and narrow ligase selection and DNA source to cell-free microbial DNA.
Stated Advantages
Enables detection of different nucleic acid forms by using adapters with different identifying sequences tied to form-preferred ligation and subsequent detection.
Supports concurrent processing of different nucleic acid forms in a sample.
Documented Applications
Cancer diagnosis and therapy tailoring by detecting mutations/genes using nucleic-acid detection methods.
Fetal health assessment for embryo/fetal genetic conditions including chromosomal aberrations and inherited disorders.
Infection assessment by distinguishing RNA versus DNA, including particle-protected/exosomal nucleic acids, and differentiating latent versus active infection states.
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