PROCESSING OF POLYNUCLEOTIDES
Methods and compositions for processing polynucleotides are provided according to some embodiments herein. In some embodiments, a sample is immobilized in a porous matrix, and non-polynucleotides are removed from the sample. In some embodiments, polynucleotides are labeled or enzymatically modified the matrix. In some embodiments, labeled or enzymatically modified polynucleotides are removed from the matrix for analysis.
1 .- 179 . (canceled)
180 . A method of processing a sample comprising a polynucleotide, the method comprising:
immobilizing the sample in a thin-layer porous matrix;
conforming the thin-layer porous matrix to a substrate;
removing non-polynucleotide molecules from the thin-layer porous matrix conformed to the substrate while the polynucleotide remains immobilized in the thin-layer porous matrix; and
at least one of:
(a) labeling the polynucleotide with a first label; or
(b) separating the polynucleotide from the thin-layer porous matrix.
181 . The method of claim 180 , wherein the polynucleotide is labeled with the first label while immobilized in the thin-layer porous matrix and subsequently separated from the thin-layer porous matrix.
182 . The method of claim 180 , wherein the polynucleotide is separated from the thin-layer porous matrix, and subsequently labeled with the first label.
183 . The method of claim 180 , wherein immobilizing the sample in the thin-layer porous matrix comprises contacting the sample with a precursor of the thin-layer porous matrix, and forming the thin-layer porous matrix from the precursor comprising the sample.
184 . The method of claim 180 , wherein the sample is immobilized in the thin-layer porous matrix after the thin-layer porous matrix has been formed from a precursor of the thin-layer porous matrix.
185 . The method of claim 180 , wherein the thin-layer porous matrix is conformed to the substrate between the substrate and another entity, thereby defining at least one of a thickness, diameter, or volume of the thin-layer porous matrix.
186 . The method of claim 180 , wherein the thin-layer porous matrix has a thickness of about 1 to 999 micrometers.
187 . The method of claim 180 , wherein the thin-layer porous matrix comprises an agarose matrix, a polyacrylamide matrix, a gelatin matrix, a collagen matrix, a fibrin matrix, a chitosan matrix, an alginate matrix, a hyaluronic acid matrix, or any combination thereof.
188 . The method of claim 180 , wherein removing non-polynucleotide molecules from the thin-layer porous matrix or porous matrix is performed in the absence of an electric field.
189 . The method of claim 180 , wherein removing non-polynucleotide molecules from the thin-layer porous matrix or porous matrix does not comprise electrophoresis.
190 . The method of claim 180 , further comprising forming the thin-layer porous matrix by cooling a matrix precursor liquid in a mold.
191 . The method of claim 180 , further comprising forming the thin-layer porous matrix by cooling a matrix precursor, wherein the substrate defines at least one surface of the thin-layer porous matrix while the matrix percursor liquid cools.
192 . The method of claim 180 , further comprising:
forming the thin-layer porous matrix by placing a precursor over a surface other than the substrate; and
moving the precursor to the substrate after the precursor cools.
193 . The method of claim 180 , wherein after the thin-layer porous matrix has been formed, the sample is added to the thin-layer porous matrix by an electromagnetic field.
194 . The method of claim 180 , wherein the thin-layer porous matrix comprises agarose and has a thickness of 100 μm to 600 μm.
195 . The method of claim 180 , wherein immobilizing the sample in a porous matrix comprises adding the sample to the thin-layer porous matrix by an electromagnetic field.
196 . The method of claim 180 , wherein the substrate comprises a mesh with openings of 0.1 to 10 nm in diameter.
197 . The method of claim 180 , wherein separating comprises electroelution.
198 . A method of processing a sample comprising a polynucleotide, the method comprising:
immobilizing the sample in a porous matrix, in an aqueous environment;
fragmenting the porous matrix comprising the immobilized sample;
removing non-polynucleotide molecules from the porous matrix while the polynucleotide remains in the porous matrix; and
separating the polynucleotide from the porous matrix.
199 . A polynucleotide preparation comprising:
a thin-layer porous matrix conformed to a substrate;
a polynucleotide immobilized in the porous matrix, wherein the polynucleotide is substantially isolated from non-polynucleotide cellular components, and wherein the polynucleotide has been site-specifically labeled or enzymatically modified while in the matrix.