Methods and compositions for nucleic acid isolation
Inventors
Baraznenok, Vera • KUTYAVIN, Alex I. • Nanassy, Oliver Z. • Sergueev, Dmitri • Gall, Alexander A.
Assignees
Publication Number
US-12365891-B2
Publication Date
2025-07-22
Expiration Date
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Abstract
Methods and compositions for isolation of nucleic acids from biological samples are provided.
Core Innovation
Methods and compositions for isolation of nucleic acids from biological samples are provided. One aspect provides a method comprising contacting a sample comprising a nucleic acid with an aqueous composition comprising a polysaccharide comprising one or more uronic acid units and concentrating the nucleic acid on a solid support thereby isolating the nucleic acid. The compositions and methods can be used to isolate, concentrate or precipitate nucleic acids from a variety of nucleic acid-containing samples.
The background identifies that preparation of nucleic acid samples prior to amplification and detection is the most challenging step of molecular diagnostics because currently available methods generally involve lengthy steps and are not easily amenable to automation. A technical feature disclosed is the use of a polysaccharide comprising one or more uronic acid units to facilitate isolation, e.g., by precipitation or flocculation, of nucleic acids from samples such as cell lysates and tissue. The inventors disclose that some polysaccharide agents, when added at concentrations ranging from about 0.1 μg/mL to about 1,000 μg/mL, facilitate recovery of nucleic acids by conventional methods with increased yields, allow isolation at room temperature compatible with automated, cartridge-based molecular diagnostics, and do not require removal of the polysaccharide agents because presence of the agents does not inhibit detection of the isolated nucleic acids by standard nucleic acid amplification methods such as PCR.
Claims Coverage
Three independent claims are identified: two directed to methods for isolation of a nucleic acid and one directed to a method for detecting a nucleic acid. The following 15 inventive features are extracted from the independent claims and their dependent recitations.
Polysaccharide comprising repeating units of Formula II
Contacting a sample comprising a nucleic acid with an aqueous composition comprising a polysaccharide comprising one or more repeating units having the structure of Formula II.
Polysaccharide further comprising repeating units of Formula III
The polysaccharide further comprises one or more repeating units of Formula (III).
Polysaccharide comprising repeating units having the structure of Formula VI
The polysaccharide comprises one or more repeating units having the structure of Formula VI.
Polysaccharide comprising repeating units having the structure of Formula V
The polysaccharide comprises one or more repeating units having the structure of Formula V.
Repeating units represented by Formula VI, VII, or VIII
The polysaccharide comprises one or more units represented by Formula VI, Formula VII, or Formula VIII.
Polysaccharide concentration from about 0.1 μg/mL to about 1000 μg/mL
The polysaccharide is present in the aqueous composition at a concentration from about 0.1 μg/mL to about 1000 μg/mL.
Polysaccharide relative molecular weight between about 120 kDa and about 500 kDa
The polysaccharide has a relative molecular weight between about 120 kDa and about 500 kDa.
Concentration of the nucleic acid by centrifugation or precipitation
The nucleic acid is concentrated by centrifugation, precipitation, or a combination thereof.
Precipitation on a solid support
The nucleic acid is concentrated by precipitation on a solid support.
Solid support comprising silica, glass, ethylenic backbone polymer, mica, polycarbonate, zeolite, or titanium dioxide
The solid support comprises a material selected from silica, glass, ethylenic backbone polymer, mica, polycarbonate, zeolite, titanium dioxide, or a combination thereof.
Solid support formats including magnetic beads and filters
The solid support is a magnetic bead, glass bead, cellulose filter, polycarbonate filter, polytetrafluoroethylene filter, polyvinylpyrrolidone filter, polyethersulfone filter, or glass filter.
Washing and eluting the nucleic acid from the solid support
The method further comprises washing and/or eluting the nucleic acid concentrated on the solid support with a wash agent, an eluting agent, or a combination thereof.
Aqueous composition further comprising lysis, chaotropic, salt, buffering, surfactant, or defoaming agents
The aqueous composition further comprises one or more of a lysis agent, a chaotropic agent, a salt, a buffering agent, a surfactant, or a defoaming agent.
Sample types including blood, plasma, serum, tissue, urine, stool, saliva, culture, or reaction mixtures
The sample comprising nucleic acid is or comprises blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, PCR reaction mixture, cell lysate preparation, or in vitro nucleic acid modification reaction mixture.
Method for detecting a nucleic acid by contacting with polysaccharide and detecting
A method for detecting a nucleic acid in a sample comprising contacting the sample with an aqueous composition comprising a polysaccharide comprising one or more repeating units having the structure of Formula II and detecting the nucleic acid.
The independent claims cover methods that employ aqueous compositions comprising defined polysaccharide repeating units (Formula II and related Formulae) to facilitate concentration or precipitation of nucleic acids onto a range of solid supports with specified concentration and molecular weight ranges, include optional compositional elements (lysis/chaotropic/salts/buffers/surfactants), encompass a breadth of solid support materials and sample types, and extend to a detecting workflow that includes contacting with the polysaccharide and detecting the nucleic acid.
Stated Advantages
Facilitates isolation, concentration, or precipitation of nucleic acids from nucleic acid-containing samples with increased yields.
Allows isolation of nucleic acids at room temperature compatible with automated, cartridge-based molecular diagnostics assays.
Does not require removal of the polysaccharide agents from the isolated nucleic acids or additional purification steps because presence of the agents does not inhibit detection by standard nucleic acid amplification methods such as PCR.
Simple and rapid methods that can be adapted to clinical laboratory automation and pre-concentration from large sample volumes for processing in microfluidic devices.
Documented Applications
Molecular diagnostic assays that utilize amplification and/or detection of nucleic acids by automated analytical techniques such as PCR, RT-PCR, qPCR, nested PCR, isothermal PCR, and other amplification methods.
Use in automated cartridge-based systems exemplified by the GeneXpert cartridge for sample extraction, amplification, and detection in a self-contained workflow.
Use of isolated nucleic acids or amplification products in hybridization protocols, nucleic acid-based microarrays, and next generation sequencing (NGS) platforms.
Kits for nucleic acid isolation comprising a polysaccharide (one or more units represented by Formulae (I)-(VIII)) optionally with lysis reagent, elution reagent, buffer, salt, or chaotropic agent components.
Isolation and detection of nucleic acids from a variety of biological samples explicitly including blood, plasma, serum, semen, tissue biopsy including paraffin-embedded tissue, urine, stool, saliva, spinal fluid, bacterial culture, mammalian cell culture, viral culture, cell lysates, PCR reaction mixtures, and in vitro nucleic acid modification reaction mixtures.
Detection of specific viral or microbial nucleic acids exemplified in the document by HBV DNA, HIV RNA, MS2 phage RNA, and fragmented Mycobacterium tuberculosis (MTB) DNA in plasma, urine, or other sample matrices.
Pre-concentration of nucleic acids from large volume samples such as plasma, urine, or water and use with magnetic bead-based extraction or filtration devices.
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