METHODS AND SYSTEMS FOR AMPLIFYING LOW CONCENTRATIONS OF NUCLEIC ACIDS
This disclosure provides methods and systems for amplifying small amounts of nucleic acids. Methods include performing amplification reactions in an emulsion format to isolate and clonally amplify discrete populations of nucleic acid molecules inside droplets. In particular, methods and systems of the invention generate an emulsion with particles that template the formation of droplets inside a tube and segregate nucleic acid molecules therein such that each droplet contains an individual nucleic acid molecule. The nucleic acid molecules are amplified inside the droplets.
1 . A method for amplifying cfDNA, the method comprising:
obtaining a sample comprising a cfDNA fragment in blood or plasma;
combining template particles and the sample in an aqueous fluid within a vessel;
adding a second fluid to the vessel;
vortexing the vessel to form a plurality of monodisperse droplets simultaneously, wherein the cfDNA fragment is isolated within one of the droplets; and
amplifying the cfDNA fragment inside the droplet.
2 . The method of claim 1 , wherein the vessel is a blood collection tube.
3 . The method of claim 1 , wherein the cfDNA fragment is ctDNA.
4 . The method of claim 3 , wherein amplifying includes generating a plurality of amplicons that can be analyzed to provide genetic information from the subject.
5 . The method of claim 4 , wherein the genetic information describes one or more mutations in the subject.
6 . The method of claim 5 , wherein the ctDNA includes a mutation specific to a tumor.
7 . The method of claim 6 , wherein the amplicons are analyzed by sequencing.
8 . The method of claim 1 , wherein amplifying the target cfDNA fragment includes barcoding the cfDNA fragment.
9 . The method of claim 1 , wherein vortexing the vessel comprises placing the vessel onto a vortexer.
10 . The method of claim 1 , wherein the template particles comprise one or more compartments.
11 . The method of claim 10 , wherein the one or more compartments contain a reagent releasable from the one or more compartments into the monodisperse droplet.
12 . The method of claim 11 , wherein the reagent is a DNA polymerase.
13 . The method of claim 12 , wherein the reagent is released from the one or more compartments in response to an external stimulus.
14 . The method of claim 13 , wherein the template particles comprise hydrogel selected from agarose, alginate, a polyethylene glycol (PEG), a polyacrylamide (PAA), acrylate, acrylamide/bisacrylamide copolymer matrix, azide-modified PEG, poly-lysine, polyethyleneimine, or any combination thereof.
15 . The method of claim 1 , wherein amplifying includes multiple displacement amplification.
16 . A method for amplifying nucleic acid molecules present at a low concentration, the method comprising:
combining template particles and nucleic acid molecules in an aqueous fluid within a vessel, wherein the concentration of the nucleic acid molecules inside the vessel is below 1 ng per μl;
adding a second fluid to the vessel;
vortexing the vessel to form a plurality of monodisperse droplets simultaneously, wherein each of the nucleic acid molecules are contained within a separate one of the droplets; and
amplifying the nucleic acid molecules contained inside the droplets.
17 . The method of claim 16 , wherein the second fluid is immiscible with the first fluid.
18 . The method of claim 17 , wherein after combining the nucleic acid molecules with the template particles the concentration of nucleic acid molecules is below 1 pg per μl.