The present invention relates to the field of pharmacogenomics and in particular to detecting the presence or absence of hypermethylated DNA. The detection of CpG methylation in marker DNA is useful for the diagnosis of cancers and the invention provides improved methods for this purpose. These improved methods allow in particular for a more sensitive detection of methylated marker DNA with high backgrounds of unmethylated marker DNA.

The present invention relates to the field of pharmacogenomics and in particular to detecting the presence or absence of hypermethylated DNA. The detection of CpG methylation in marker DNA is useful for the diagnosis of cancers and the invention provides improved methods for this purpose. These improved methods allow in particular for a more sensitive detection of methylated marker DNA with high backgrounds of unmethylated marker DNA.

A method is provided that includes adding, using a polymerase coupled to a substrate, nucleotides in a solution to a first polynucleotide using at least a sequence of a second polynucleotide. The method includes separating, using labels respectively coupled to the nucleotides, quenchers from respective fluorophores. The method includes then detecting a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least fluorescence from the respective fluorophores.

A method is provided that includes adding, using a polymerase coupled to a substrate, nucleotides in a solution to a first polynucleotide using at least a sequence of a second polynucleotide. The method includes separating, using labels respectively coupled to the nucleotides, quenchers from respective fluorophores. The method includes then detecting a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least fluorescence from the respective fluorophores.

A method for identifying a nucleotide in a template nucleic acid by (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have an extendable 3′ end; (b) contacting the plurality with: (i) blocked nucleotides to produce a first subset of the primer-template nucleic acid hybrids that include a blocked nucleotide at the 3′ end, and (ii) a ternary complex inhibitor to produce a second subset of the primer-template nucleic acid hybrids that include a ternary complex inhibitor; (c) forming ternary complexes that each include a polymerase, a primer-template nucleic acid hybrid of the first subset, and a cognate nucleotide; and (d) detecting the ternary complexes, thereby identifying a nucleotide in the template nucleic acid.

According to one embodiment, a method for detecting target nucleic acid includes the following steps. (A) A reaction field is formed by placing a reaction mixture on an electrode, and the reaction mixture contains the sample, a primer set, an amplification enzyme, 4 mM to 30 mM of magnesium ion, and a redox probe. The redox probe has an oxidation reduction potential, which generates an electric signal of which amplitude increases. (B) The reaction field is maintained under an amplification reaction condition. (C) The electric signal is detected with the electrode. (D) Existence or quantity of the target nucleic acid is determined.

The invention relates to a set of oligonucleotide primers and their use in methods for amplifying and detecting oligonucleotides, detecting pathogens, or diagnosing infections such as SARS-CoV-2 and Covid-19. The primer set includes a switch oligonucleotide that is adapted to anneal to a forward or reverse primer of the set at temperatures that are below the temperature range for amplification of the DNA. The switch oligonucleotide prevents amplification from the complementary primer when the complementary primer is bound to the switch oligonucleotide. The invention also provides a method of reducing false positives in the detection of a target DNA or RNA sequence using loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP), by using such a switch oligonucleotide.

The invention relates to a set of oligonucleotide primers and their use in methods for amplifying and detecting oligonucleotides, detecting pathogens, or diagnosing infections such as SARS-CoV-2 and Covid-19. The primer set includes a switch oligonucleotide that is adapted to anneal to a forward or reverse primer of the set at temperatures that are below the temperature range for amplification of the DNA. The switch oligonucleotide prevents amplification from the complementary primer when the complementary primer is bound to the switch oligonucleotide. The invention also provides a method of reducing false positives in the detection of a target DNA or RNA sequence using loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP), by using such a switch oligonucleotide.

The present invention provides methods, compositions and kits for enriching and determining nucleotide sequences of a plurality of target loci from a sample comprising nucleic acids. The methods comprise one or more cycles of primer extension followed by PCR amplification of target sequences using nested target-specific primers.

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.