Provided herein are reagents and methods for comprehensively enriching potential variants within targeted regions, named Amplicon Comprehensive Enrichment (ACE). The sequence variants enriched can include single nucleotide polymorphisms (SNPs), single nucleotide variants, or small insertions and deletions. Embodiments include procedures for integration with real-time polymerase chain reaction, next generation sequencing (NGS), and long-read sequencing.

Provided herein are methods and kits for discriminating a target sequence from a reference sequence in a polymerase chain reaction (PCR) by use of a promiscuity-blocking nucleotide juror (PBNJ) oligonucleotide.

Provided herein are methods and kits for discriminating a target sequence from a reference sequence in a polymerase chain reaction (PCR) by use of a promiscuity-blocking nucleotide juror (PBNJ) oligonucleotide.

The present invention relates to a method for analyzing the presence or absence of aneuploidy of a target chromosome with high sensitivity, and a composition for detecting chromosomal aneuploidy, and more particularly to a method of identifying chromosomal aneuploidy by amplifying a control nucleotide sequence, located on a chromosome not associated with chromosomal aneuploidy, and a target nucleotide sequence located on a chromosome associated with chromosomal aneuploidy, by using the same primer, and then hybridizing the amplification products with an assay probe that differs by one or two nucleotides from the control nucleotide sequence and with an elimination probe that comprises part or all of a sequence of the assay probe, which hybridizes with the target nucleotide sequence or the control nucleotide sequence, the elimination probe having a higher binding affinity for the amplification products than the assay probe, and analyzing melting curves of the hybridization products. The method for detecting chromosomal aneuploidy according to the present invention may analyze the ratio of the target nucleotide sequence to the control nucleotide sequence at high resolution by eliminating equal amounts (certain proportions) of the target nucleotide sequence and the control nucleotide sequence from the analysis using the elimination sequence. This method is useful because numerical abnormalities (aneuploidy) in chromosomes (e.g., fetal chromosomes in maternal blood, and circulating tumor DNA in cancer patients) present at low rates can be detected quickly with high sensitivity by the use of this method.

The present invention relates to a method for analyzing the presence or absence of aneuploidy of a target chromosome with high sensitivity, and a composition for detecting chromosomal aneuploidy, and more particularly to a method of identifying chromosomal aneuploidy by amplifying a control nucleotide sequence, located on a chromosome not associated with chromosomal aneuploidy, and a target nucleotide sequence located on a chromosome associated with chromosomal aneuploidy, by using the same primer, and then hybridizing the amplification products with an assay probe that differs by one or two nucleotides from the control nucleotide sequence and with an elimination probe that comprises part or all of a sequence of the assay probe, which hybridizes with the target nucleotide sequence or the control nucleotide sequence, the elimination probe having a higher binding affinity for the amplification products than the assay probe, and analyzing melting curves of the hybridization products. The method for detecting chromosomal aneuploidy according to the present invention may analyze the ratio of the target nucleotide sequence to the control nucleotide sequence at high resolution by eliminating equal amounts (certain proportions) of the target nucleotide sequence and the control nucleotide sequence from the analysis using the elimination sequence. This method is useful because numerical abnormalities (aneuploidy) in chromosomes (e.g., fetal chromosomes in maternal blood, and circulating tumor DNA in cancer patients) present at low rates can be detected quickly with high sensitivity by the use of this method.

Methods for enhancing specificity of an analyte binding moiety or probe oligonucleotide to an analyte are provided herein. For example, methods provided herein include blocking a capture binding domain, thereby preventing hybridization to the capture domain of the capture probe affixed to a substrate. Further methods include releasing the block from the capture binding domain, thereby allowing the capture binding domain to specifically bind to the capture domain of the capture probe on the substrate.

Methods for enhancing specificity of an analyte binding moiety or probe oligonucleotide to an analyte are provided herein. For example, methods provided herein include blocking a capture binding domain, thereby preventing hybridization to the capture domain of the capture probe affixed to a substrate. Further methods include releasing the block from the capture binding domain, thereby allowing the capture binding domain to specifically bind to the capture domain of the capture probe on the substrate.

A depleted sequencing library can be prepared by providing a composition comprising a heterogeneous mixture of linear nucleic acids having a first terminus and a second terminus. A first subset of target nucleic acids and a second subset of non-target nucleic acids can include a first adaptor region at the first terminus and a second adaptor region at the second terminus. A third subset of the target nucleic acids and a fourth subset of the non-target nucleic acids include the second adaptor region at the first terminus and at the second terminus. Removable blocker oligonucleotides can be added to the composition, non-target nucleic acids can be removed from the composition by sequence capture to bait oligonucleotides, and the composition can be treated to reduce a quantity of free blocker oligonucleotides that are not annealed to an adaptor sequence or to a sequence substantially complementary to an adaptor sequence.

A depleted sequencing library can be prepared by providing a composition comprising a heterogeneous mixture of linear nucleic acids having a first terminus and a second terminus. A first subset of target nucleic acids and a second subset of non-target nucleic acids can include a first adaptor region at the first terminus and a second adaptor region at the second terminus. A third subset of the target nucleic acids and a fourth subset of the non-target nucleic acids include the second adaptor region at the first terminus and at the second terminus. Removable blocker oligonucleotides can be added to the composition, non-target nucleic acids can be removed from the composition by sequence capture to bait oligonucleotides, and the composition can be treated to reduce a quantity of free blocker oligonucleotides that are not annealed to an adaptor sequence or to a sequence substantially complementary to an adaptor sequence.

Provided herein are methods of determining a location of a target analyte in a non-permeabilized biological sample that include the use of a blocking probe.