Isolation of nucleic acids
Provided herein is technology relating to isolating nucleic acids. In particular, the technology relates to methods and kits for extracting nucleic acids from problematic samples such as stool.
1. A method for partitioning a human stool homogenate comprising human DNA and bacterial DNA to produce a human target DNA preparation and a residual supernatant, the method comprising:
a) treating a fluid fraction from a human stool homogenate with insoluble polyvinylpyrrolidone to bind inhibitor, if present, in an insoluble inhibitor complex, to produce a treated stool fluid;
b) exposing the treated stool fluid to a target sequence-specific capture reagent comprising particles attached to oligonucleotides complementary to at least a portion of first human target DNAs under conditions wherein the first target sequence-specific capture reagent forms first capture complexes comprising the target sequence-specific capture reagent and the first human target DNAs;
c) separating the first capture complexes from the treated stool fluid to produce:
i) a human target DNA preparation comprising the first human target DNAs captured in first capture complexes in step b); and
ii) a residual supernatant comprising bacterial DNA and human target DNAs not complementary to the oligonucleotides of the target-sequence specific capture reagent used in step b).
2. The method of claim 1 , wherein the human target DNA preparation is a solution comprising first human target DNAs recovered from the first capture complexes.
3. The method of claim 1 , wherein the human target DNA preparation comprises a collection of the particles attached to oligonucleotides complementary to at least a portion of the first human target DNAs.
4. The method of claim 1 , wherein the first human target DNAs comprise at least one human target DNA selected from NDRG4, BMP3, and KRAS DNAs.
5. The method of claim 4 , wherein the first human target DNAs comprise NDRG4, BMP3, and KRAS DNAs.
6. The method of claim 1 , wherein said first target human DNAs comprise a reference gene.
7. The method of claim 6 , wherein the reference gene is β-actin.
8. The method of claim 1 , further comprising recovering human target DNAs from the first capture complexes to produce a human target DNA preparation that is a solution comprising the first human target DNAs.
9. The method of claim 1 , further comprising:
d) exposing the residual supernatant to a further target sequence-specific capture reagent comprising particles attached to oligonucleotides complementary to at least a portion of second human target DNAs under conditions wherein the further target sequence-specific capture reagent forms second capture complexes comprising the further target sequence-specific capture reagent and the second human target DNAs;
e) separating the second capture complexes from the residual supernatant to produce:
i) a human target DNA preparation comprising the second human target DNAs captured in the second capture complexes in step d); and
ii) a residual supernatant comprising bacterial DNA and human target DNAs not complementary to the oligonucleotides of the target-sequence specific capture reagent used in step d).
10. The method of claim 1 , wherein treating the fluid fraction from the human stool homogenate with insoluble polyvinylpyrrolidone to produce said treated stool fluid comprises passing treated stool fluid through porous filtering material, wherein the insoluble inhibitor complex is retained by the porous filtering material.
11. The method of claim 1 , wherein the particle of said target-sequence specific capture reagent comprises a magnetic particle, and wherein separating the first capture complexes comprises exposing the first capture complexes to a magnetic field.