Quantitative nuclease protection assay (QNPA) and sequencing (QNPS) improvements
DCFirst Claim
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1. A method of determining a sequence of at least one target nucleic acid molecule in a sample, comprising:
- contacting the sample with at least one nuclease protection probe comprising a flanking sequence (NPPF) under conditions sufficient for the NPPF to specifically bind to the target nucleic acid molecule,wherein the NPPF comprises;
a 5′
-end and a 3′
-end,a sequence complementary to a region of the target nucleic acid molecule, permitting specific binding between the NPPF and the target nucleic acid molecule,a flanking sequence located 5′
, 3′
, or both, to the sequence complementary to the target nucleic acid molecule, wherein the flanking sequence comprises at least 12 contiguous nucleotides not found in a nucleic acid molecule present in the sample, providing a universal amplification sequence, and wherein the flanking sequence is complementary to at least a portion of an amplification primer;
contacting the sample with a nucleic acid molecule comprising a sequence complementary to the flanking sequence (CFS) under conditions sufficient for the flanking sequence to specifically bind to the CFS;
contacting the sample with a nuclease specific for single-stranded nucleic acid molecules under conditions sufficient to remove unbound nucleic acid molecules, thereby generating a digested sample comprising NPPFs hybridized to the target nucleic acid molecule and to the CFS(s);
amplifying NPPFs in the digested sample with the amplification primer, thereby generating NPPF amplicons; and
sequencing at least a portion of the NPPF amplicons, thereby determining the sequence of the at least one target nucleic acid molecule in the sample.
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Abstract
The present disclosure provides an improvement to quantitative Nuclease Protection Assay (qNPA) and quantitative Nuclease Protection Sequencing (qNPS) methods. The disclosed methods use nuclease protection probes (NPPs) that include 5′-end and/or 3-end flanking sequences, which provide a universal hybridization and/or amplification sequence. The disclosed methods can be used to sequence or detect target nucleic acid molecules, such as those present in fixed or insoluble samples.
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Citations
20 Claims
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1. A method of determining a sequence of at least one target nucleic acid molecule in a sample, comprising:
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contacting the sample with at least one nuclease protection probe comprising a flanking sequence (NPPF) under conditions sufficient for the NPPF to specifically bind to the target nucleic acid molecule, wherein the NPPF comprises; a 5′
-end and a 3′
-end,a sequence complementary to a region of the target nucleic acid molecule, permitting specific binding between the NPPF and the target nucleic acid molecule, a flanking sequence located 5′
, 3′
, or both, to the sequence complementary to the target nucleic acid molecule, wherein the flanking sequence comprises at least 12 contiguous nucleotides not found in a nucleic acid molecule present in the sample, providing a universal amplification sequence, and wherein the flanking sequence is complementary to at least a portion of an amplification primer;contacting the sample with a nucleic acid molecule comprising a sequence complementary to the flanking sequence (CFS) under conditions sufficient for the flanking sequence to specifically bind to the CFS; contacting the sample with a nuclease specific for single-stranded nucleic acid molecules under conditions sufficient to remove unbound nucleic acid molecules, thereby generating a digested sample comprising NPPFs hybridized to the target nucleic acid molecule and to the CFS(s); amplifying NPPFs in the digested sample with the amplification primer, thereby generating NPPF amplicons; and sequencing at least a portion of the NPPF amplicons, thereby determining the sequence of the at least one target nucleic acid molecule in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification