Method for separating genomic dna for amplification of short nucleic acid targets
AssigneeBecton, Dickinson and Company
InventorsHonghua Zhang, Daniel Jacob Koslover
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Abstract
Disclosed herein include methods, compositions, and kits for use in detecting a target nucleic acid sequence in a sample. Provided include acidic compositions for separating dsDNA. In some embodiments, the acidic compositions comprise a monovalent salt and/or a divalent salt, one or more surfactants, and an acidic agent. In some embodiments, the pH of the acidic composition is less than 4. Also provided herein are reagent compositions comprising a buffering agent and one or more amplification agents.
Claims85
A method for amplifying a target nucleic acid sequence in a sample, comprising:
- (a) contacting a sample comprising biological entities with an acidic composition to generate an acidic mixture,
- wherein the acidic composition is capable of lysing the biological entities to release sample nucleic acids comprised therein, wherein the sample nucleic acids comprise double-stranded DNA (dsDNA) suspected of comprising a target nucleic acid sequence, wherein the target nucleic acid sequence is no longer than 100 nucleotides in length,
- wherein the acidic composition has a pH of less than 4 and comprises (i) a monovalent salt and/or a divalent salt, (ii) one or more surfactants, and (iii) an acidic agent, thereby denaturing the dsDNA to generate single-stranded DNA (ssDNA);
- (b) contacting a reagent composition comprising a buffering agent with the acidic mixture to generate a neutral mixture, wherein the neutral mixture comprises the ssDNA, wherein the reagent composition comprises one or more amplification reagents;
- and
- (c) amplifying the target nucleic acid sequence in the neutral mixture, thereby generating a nucleic acid amplification product, optionally the reagent composition is a dried composition or a wet composition.
An acidic composition for lysing biological entities and denaturing double-stranded DNA (dsDNA) comprised therein, comprising:
- a monovalent salt at a concentration of less than 30 mM in the acidic composition and/or a divalent salt at a concentration of less than 15 mM in the acidic composition;
- one or more surfactants;
- and
- an acidic agent present in the acidic composition at a concentration of less than 100 mM, and
- wherein the acidic composition has a pH less than 4.
Dependencies are inferred from the claim text.