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 amplifying a human target nucleic acid from a fluid fraction prepared from human stool homogenate, the method comprising:
i) 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;
ii) exposing the treated stool fluid to a target sequence-specific capture reagent comprising particles attached to oligonucleotides complementary to at least a portion of human target nucleic acid, under conditions wherein the target sequence-specific capture reagent forms target sequence-specific capture reagent/human target nucleic acid capture complexes;
iii) separating the capture complexes from the treated stool fluid;
iv) recovering human target nucleic acid from the separated capture complexes of step iii) in a human target nucleic acid solution; and
v) treating a portion of the human target nucleic acid solution in an amplification reaction mixture comprising DNA polymerase, wherein the amplification reaction mixture comprises primers for amplifying a region of a human target nucleic acid captured by the target sequence-specific capture reagent.
2. The method of claim 1 , wherein the portion of human target nucleic acid solution has a volume and the amplification reaction mixture has a total volume, and wherein the volume of the human target nucleic acid solution is at least 1/25 th of the total volume of the amplification reaction mixture.
3. The method of claim 1 , wherein the portion of human target nucleic acid solution has a volume and the amplification reaction mixture has a total volume, and wherein the volume of the human target nucleic acid solution is at least ⅕ th of the total volume of the amplification reaction mixture.
4. The method of claim 1 , wherein the portion of human target nucleic acid solution has a volume and the amplification reaction mixture has a total volume, and wherein the volume of the human target nucleic acid solution is at least ⅓rd of the total volume of the amplification reaction mixture.
5. The method of claim 1 , wherein the amplification reaction mixture further comprises a detection probe complementary to at least a portion of DNA amplified from the region of human target nucleic acid captured by the target sequence-specific capture reagent.
6. The method of claim 5 , wherein the amplification reaction mixture further comprises a flap endonuclease.
7. The method of claim 1 , wherein treating the fluid fraction from a human stool homogenate with insoluble polyvinylpyrrolidone to produce the treated stool fluid comprises passing treated stool fluid through porous filtering material, wherein the insoluble inhibitor complex is retained by the porous filtering material.
8. The method of claim 1 , wherein particles are magnetic particles and wherein separating the capture complexes comprises exposing the capture complexes to a magnetic field.
9. The method of claim 1 , wherein recovering the human target nucleic acid comprises eluting the human target nucleic acid from the separated capture complexes.
10. The method of claim 1 , wherein the human target nucleic acid captured by the target sequence-specific capture reagent comprises DNA from a gene associated with colorectal cancer and/or colorectal adenoma.
11. The method of claim 1 , wherein the human target nucleic acid captured by the target sequence-specific capture reagent comprises at least one of NDRG4, BMP3, and KRAS DNA.
12. The method of claim 1 , wherein the human target nucleic acid comprises a human reference gene.
13. The method of claim 12 , wherein the human reference gene is β-actin.
14. The method of claim 1 , wherein the human target nucleic acid is DNA.