Enrichment of nucleic acids by complimentary capture
Assays can be used to detect mutations found in neoplasms of the pancreas, as well as for other neoplasms and other uses. Nucleic acids can be captured from body fluids such as cyst fluids. Thousands of oligonucleotides can be synthesized in parallel, amplified and ligated together. The ligated products can be further amplified. The amplified, ligated products are used to capture complementary DNA sequences, which can be analyzed, for example by massively parallel sequencing.
1. A method for enriching a sample comprising nucleic acids for target nucleic acid analytes, comprising the steps of:
synthesizing a set of probes for one or more analytes of interest, wherein the probes are complementary to plus or minus strands of a target nucleic acid analyte, wherein each probe has a 5′ universal priming site and a 3′ universal priming site;
amplifying the set of probes using primers complementary to the 5′ universal priming site and primers complementary to the 3′ universal priming site;
ligating the amplified probes to form double-stranded concatamers;
isothermally amplifying the double-stranded concatamers to form single-stranded amplified concatamers;
binding the single-stranded amplified concatamers to a solid support;
contacting the solid support with the sample comprising nucleic acids under hybridization conditions so that complementary nucleic acids in the sample are captured on the solid support and non-complementary nucleic acids are removed;
eluting captured nucleic acids from the solid support.
2. A method for enriching a sample for target nucleic acid analytes, comprising the steps of:
synthesizing a set of probes for one or more analytes of interest, wherein the probes are complementary to plus or minus strands of a target nucleic acid analyte, wherein each probe has a 5′ universal priming site and a 3′ universal priming site;
amplifying the set of probes using primers complementary to the 5′ universal priming site and primers complementary to the 3′ universal priming site;
ligating the amplified probes to form double-stranded concatamers;
isothermally amplifying the double-stranded concatamers in the presence of biotinylated nucleotides, such that biotinylated nucleotides are incorporated into amplified, single-stranded concatamers;
contacting the amplified, single-stranded concatamers with the sample nucleic acids to form a mixture, and subjecting the mixture to hybridization conditions so that complementary nucleic acids in the mixture hybridize to the amplified, single-stranded concatamers;
contacting the mixture with a solid support so that hybridized nucleic acids are captured on the solid support, wherein the solid support comprises avidin or streptavidin;
washing the solid support to remove nucleic acids which do not comprise biotin;
eluting captured nucleic acids from the solid support.
3. The method of claim 1 further comprising the step of: analyzing nucleotide sequence of the eluted, captured nucleic acids.
4. The method of claim 3 wherein the analyzing is massively parallel.
5. The method of claim 2 further comprising: analyzing nucleotide sequence of the eluted, captured nucleic acids.
6. The method of claim 5 wherein the analyzing is massively parallel.
7. The method of claim 1 further comprising the step of: amplifying the eluted, captured nucleic acids.
8. The method of claim 2 further comprising the step of: amplifying the eluted, captured nucleic acids.
9. The method of claim 1 wherein the set of probes are synthesized on a solid array.
10. The method of claim 9 wherein the set of probes are cleaved from the solid array to form an aqueous solution comprising the set of probes.
11. The method of claim 2 wherein the set of probes are synthesized on a solid array.
12. The method of claim 11 wherein the set of probes are cleaved from the solid array to form an aqueous solution comprising the set of probes.
13. The method of claim 1 wherein the probes are complementary to both plus and minus strands of a target nucleic acid analyte.
14. The method of claim 1 wherein probes for one analyte overlap with a register of three nucleotide displacement.
15. The method of claim 13 wherein probes for one analyte overlap with a register of three nucleotide displacement.
16. The method of claim 1 wherein the solid support is a filter.
17. The method of claim 1 wherein the solid support is a bead.
18. The method of claim 2 wherein the probes are complementary to both plus and minus strands of a target nucleic acid analyte.
19. The method of claim 2 wherein probes for one analyte overlap with a register of three nucleotide displacement.
20. The method of claim 18 wherein probes for one analyte overlap with a register of three nucleotide displacement.
21. The method of claim 2 wherein the solid support is a filter.
22. The method of claim 2 wherein the solid support is a bead.