COMPOSITIONS AND METHODS FOR NUCLEIC ACID NORMALIZATION
Methods, compositions, and kits of the present disclosure for normalizing a mass amount of nucleic acids in each of a plurality of test samples are provided, for use in methods of nucleic acid analysis, such that substantially equal amounts of nucleic acids are present in the samples to be analyzed.
1 . A method of normalizing a mass amount of nucleic acids in each of a plurality of test samples, comprising:
providing a plurality of input samples containing nucleic acids in an aqueous liquid, each of the plurality of input samples present in a separate container;
adding a binding mixture to each container, producing a normalization mixture in each container, the binding mixture comprising:
a quantity of magnetic particles comprising pendant hydroxyl functional groups,
a chelating agent,
a binding buffer, and
an alcohol,
wherein the binding buffer comprises a buffered aqueous solution of a chaotrope, wherein the quantity of magnetic particles is capable of reversibly and non-specifically binding nucleic acids at a binding capacity in the range of about 1 nanogram to about 5 micrograms,
and wherein each of the plurality of input samples contains a mass amount of nucleic acids greater than the binding capacity of the quantity of magnetic particles;
incubating each normalization mixture under binding conditions, thereby reversibly and non-specifically binding a portion of the nucleic acids to the magnetic particles;
separating the magnetic particles with the reversibly and non-specifically bound nucleic acids from non-bound nucleic acids by application of a magnetic field; and
eluting the reversibly and non-specifically bound nucleic acids from the magnetic particles, producing a plurality of test samples, wherein each of the plurality of test samples comprises isolated nucleic acids having a mass amount, wherein the mass amount of the isolated nucleic acids is approximately equivalent to the binding capacity of the magnetic particles, thereby providing a normalized mass amount of nucleic acids in each of the plurality of test samples.
2 . The method of claim 1 , wherein no internal standard is added to the container.
3 . The method of claim 1 , wherein the mass amount of nucleic acids in the input samples is not quantitated.
4 . The method of claim 1 , further comprising pooling the plurality of test samples, producing pooled test samples, and sequencing at least some of the nucleic acids in the pooled test samples.
5 . The method of claim 1 , wherein the chaotrope comprises one or more of: urea, guanidinium bromide (guanidine hydrobromide or guanidine monohydrobromide), guanidinium iodide (guanidine hydroiodide), guanidinium chloride (guanidine hydrochloride), guanidine thiocyanate (guanidinium thiocyanate), guanidine nitrate (guanidinium nitrate), guanidine sulfate salt (guanidinium sulfate), guanidine carbonate salt (guanidinium carbonate), sodium iodide, and sodium perchlorate.
6 . The method of claim 1 , wherein the binding buffer does not include PEG.
7 . The method of claim 1 , wherein the magnetic particles do not comprise carboxyl functional moieties and/or amine functional moieties.
8 . The method of claim 1 , wherein the magnetic particles comprising pendant hydroxyl functional groups comprise a hydroxyl-functionalized spacer covalently bonded to, and extending from the magnetic particle and/or a coating of the magnetic particles, wherein the spacer comprises a chain of at least 3 atoms covalently bonded to a hydroxyl functional group.
9 . The method of claim 1 , wherein the chelating agent is one or more of: diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA).
10 . The method of claim 1 , wherein the alcohol is one or more of: methanol, ethanol and isopropanol.
11 . The method of claim 1 , wherein the binding buffer comprises a buffered aqueous solution of guanidine hydrochloride and potassium acetate or sodium acetate, and has a pH in the range of about pH 4 to about pH 6.
12 . The method of claim 1 , further comprising recovering the non-bound nucleic acids of one or more normalization mixtures.
13 . The method of claim 1 , comprising transferring the non-bound nucleic acids of one or more normalization mixtures to corresponding containers;
adding a binding mixture to each corresponding container, producing a recovery mixture in each corresponding container, the binding mixture comprising:
a quantity of magnetic particles comprising pendant hydroxyl functional groups, a chelating agent,
a binding buffer, and
an alcohol,
wherein the binding buffer comprises a buffered aqueous solution of a chaotrope,
wherein the quantity of magnetic particles is capable of reversibly and non-specifically binding nucleic acids at a binding capacity in the range of about 1 nanogram to about 5 micrograms,
and wherein each of the recovery mixtures contains a mass amount of nucleic acids greater than, less than, or equal to, the binding capacity of the quantity of magnetic particles;
incubating each recovery mixture under binding conditions, thereby reversibly and non-specifically binding all, or a portion of, the nucleic acids to the magnetic particles, producing magnetic particles reversibly and non-specifically bound to the recovered nucleic acids;
separating the magnetic particles reversibly and non-specifically bound to the recovered nucleic acids by application of a magnetic field; and
eluting the reversibly and non-specifically bound recovered nucleic acids from the magnetic particles.
14 . The method of claim 1 , wherein the volume:volume ratio of the alcohol to the binding buffer is in the range of about 0.25:1 to about 1.75:1.
15 . A kit, comprising:
magnetic particles comprising pendant hydroxyl functional groups;
a binding buffer comprising a buffered aqueous solution of a chaotrope;
a wash liquid; and
an elution buffer.
16 . The kit of claim 15 , further comprising a chelating agent and an alcohol.
17 . The kit of claim 15 , wherein the binding buffer comprises a buffered aqueous solution of guanidine hydrochloride and potassium acetate or sodium acetate, and has a pH in the range of about pH 4 to about pH 6.
18 . The kit of claim 15 , wherein the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA.
19 . The kit of claim 15 , wherein the wash liquid comprises about 60% to about 100% ethanol.
20 . The kit of claim 15 , wherein the magnetic particles comprising pendant hydroxyl functional groups comprise a hydroxyl-functionalized spacer covalently bonded to, and extending from the magnetic particle and/or a coating of the magnetic particle, wherein the spacer comprises a chain of at least 3 atoms covalently bonded to a hydroxyl functional group.