Enrichment of Nucleic Acids by Complimentary Capture
AssigneeThe Johns Hopkins University
InventorsBert Vogelstein, Kenneth W. Kinzler, Nickolas Papadopoulos, Jian WU
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Abstract
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.
Claims22
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 common 5′ and 3′ universal priming site;
- amplifying the set of probes using primers complementary to the universal priming sites;
- ligating the amplified probes to form concatamers;
- isothermally amplifying the concatamers;
- binding the 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.
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 common 5′ and 3′ universal priming site;
- amplifying the set of probes using primers complementary to the universal priming sites;
- ligating the amplified probes to form concatamers;
- isothermally amplifying the concatamers in the presence of biotyinylated nucleotides, such that biotinylated nucleotides are incorporated into the concatamers;
- contacting the amplified 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 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.
Dependencies are inferred from the claim text.