The present invention discloses a method for detecting the mutation and methylation of tumor-specific genes in ctDNA, and this method can simultaneously detect the mutation (including point mutation, insertion-deletion mutation, HBV integration and other mutation forms) and/or methylation of tumor-specific genes in ctDNA in one sample. Not only the sample size requirement is low, but the MC library prepared by this method can support 10-20 subsequent detections. The results of each test can represent the mutation status of all the original ctDNA specimens and the methylation modification status of the region covered by the restriction sites, without reducing the sensitivity and specificity. The present invention has important clinical significance for early tumor screening, disease tracking, efficacy evaluation, prognosis prediction and the like, and has great application value.

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

The present disclosure is directed to a polynucleotide capable of signaling under preselected conditions. For example, the present disclosure relates to a method of reconfiguring a nucleic acid or polynucleotide, including: contacting a deoxyribonucleic acid (DNA) nanoswitch and nucleic acid to form a DNA nanoswitch-nucleic acid complex having a first conformation, wherein the first conformation is characterized as locked; contacting the DNA nanoswitch-nucleic acid complex with a biological specimen to form a mixture, wherein when the nucleic acid is ribonucleic acid (RNA) and the biological specimen includes one or more ribonucleases, the first conformation changes to a second conformation characterized as open; processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal.

The present disclosure is directed to a polynucleotide capable of signaling under preselected conditions. For example, the present disclosure relates to a method of reconfiguring a nucleic acid or polynucleotide, including: contacting a deoxyribonucleic acid (DNA) nanoswitch and nucleic acid to form a DNA nanoswitch-nucleic acid complex having a first conformation, wherein the first conformation is characterized as locked; contacting the DNA nanoswitch-nucleic acid complex with a biological specimen to form a mixture, wherein when the nucleic acid is ribonucleic acid (RNA) and the biological specimen includes one or more ribonucleases, the first conformation changes to a second conformation characterized as open; processing the mixture under conditions sufficient to separate the first conformation, and when present, the second conformation; and reacting the first conformation, and when present, the second conformation with an indicator under conditions sufficient to form a signal.

A method for identifying a nucleic acid template that includes (a) providing a plurality of primer-template hybrids, wherein a first hybrid of the plurality includes a first template hybridized to a first primer, and wherein a second hybrid of the plurality includes a second template hybridized to a second primer, the second primer having a ternary complex inhibitor moiety at the 3′ end; (b) delivering polymerases and nucleotides to the plurality, whereby the first hybrid binds a polymerase and nucleotide to form a stabilized ternary complex and whereby the second hybrid does not bind a polymerase and nucleotide to form a stabilized ternary complex; and (c) detecting the stabilized ternary complex to identify the first template.

This invention allows ultra-low levels of virtually any biological analyte to be detected and quantified rapidly, simply and inexpensively with an electrochemical biosensor using a novel electrochemically detectable tag that replaces optical labels. The tag binds to an analyte directly, or indirectly using one or more ligands and particles, and consists of a quadruplex electrochemically detectable oligonucleotide rich in guanine, or a single-stranded electrochemically detectable oligonucleotide rich in guanine that self-assembles into a quadruplex electrochemically detectable oligonucleotide when exposed to cations that enable quadruplex self-assembly. Quadruplex electrochemically detectable oligonucleotide tags are exposed, adsorbed or hybridized at the surface of a biosensor working electrode. An electrochemical technique facilitates the quadruplex tags to produce 8-oxoguanine oxidation signals proportional to the analyte level in the samples. The resulting analyte levels measured from 8-oxoguanine oxidation signals are 1,000 times lower than analyte levels measured from guanine oxidation signals.

Bistable devices are constructed using a polynucleotide platform for the sensing of molecular events such as binding or conformational changes of target molecules. Uses include measurement of target concentration, measuring the effect of environmental condition (such as heat, light, or pH) on the target, or screening a library for molecules that bind the target or modulate its biological function. Devices comprise three regions: a top lid, bottom lid, and flexible linker or hinge between them. A device has an open configuration in which the top and bottom lid are separated, and a closed configuration they are bound close together. Binding domains or variations of the target molecule are fixed to a device so that when the molecular event occurs, the device switches from open to closed, or vice versa, which generates a signal. Devices carry DNA tags to enable separation of open and closed devices, as well as barcoding for multiplexed detection.

An RNA tagging system for visualization of single mRNA molecules based on a MSB-MCP system, as well as methods of use.

An RNA tagging system for visualization of single mRNA molecules based on a MSB-MCP system, as well as methods of use.