Isolation of nucleic acids
View Patent ↗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 system for isolating a target human DNA from a human stool sample, the system comprising:
a) a volume of stool homogenization solution suitable for processing a human stool sample having a mass of at least 4 grams;
b) an inhibitor-adsorbing composition comprising insoluble polyvinylpolypyrrolidone particles;
c) a spin filter, comprising
i) a hollow body;
ii) a bottom end; and
iii) an open top end opposite the bottom end,
wherein the hollow body of said spin filter is made from a porous filtering material;
d) guanidine thiocyanate; and
e) a plurality of different magnetic target-specific capture particles, wherein different target-specific capture particles comprise covalently-attached oligonucleotides complementary to at least a portion of different target human DNAs, and wherein said different target human DNAs comprise NDRG4, BMP3, and KRAS.
2. The system of claim 1 , further comprising a magnet.
3. The system of claim 1 wherein said polyvinylpolypyrrolidone is in a premeasured form.
4. The system of claim 1 wherein said polyvinylpolypyrrolidone is provided as a capsule or pressed tablet comprising a premeasured amount of polyvinylpolypyrrolidone.
5. The system of claim 1 , further comprising an elution solution and/or a wash solution.
6. The system of claim 1 , further comprising a sample container configured to hold said stool sample.
7. The system of claim 1 further comprising a vessel in which to hold isolated target human DNA.
8. The system of claim 1 , further comprising a shipping container.
9. The system of claim 1 , further comprising a control reagent.
10. The system of claim 9 , wherein said control comprises a nucleic acid.
11. The system of claim 1 , wherein said covalently-attached oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOS:2-3 and 6-7.
12. The system of claim 1 , further comprising a magnetic target-specific capture particle comprising an oligonucleotide that is complementary to at least a portion of a human reference DNA.
13. The system of claim 1 , plurality of target human DNAs further comprises vimentin, TFPI2, or beta-actin.
14. The system of claim 13 , wherein said covalently-attached oligonucleotides comprise sequences selected from the group consisting of SEQ ID NOS:1-7.
15. The system of claim 12 , wherein said human reference DNA is beta-actin.
16. The system of claim 15 , wherein said covalently-attached oligonucleotides comprise the sequence of SEQ ID NO:5.