The present invention relates to methods for detecting a chromosomal aneuploidy in a foetus carried by a pregnant female. Such methods are based on one or more of particular configurations and/or detections and/or analyses of two or more regions of DNA, including those that show differential methylation between DNA that originates from cells of a foetus (and/or the placenta of a foetus) and DNA of maternal origin. Such methods utilise a sample taken from said pregnant female, which sample comprises DNA that originates from cells of a foetus and/or the placenta of a foetus in admixture with differently methylated DNA of maternal origin. Such methods have diagnostic, prognostic and/or predictive utility; in particular for the detection/diagnosis of chromosomal aneuploidy, such as a trisomy, in a foetus, and/or for detecting an increased risk of a pregnant female suffering from or developing a pregnancy-associated medical condition. The present invention also relates to compositions, kits, computer program products and other aspects that may be used in, useful for or related to the practice of such methods.

A method of identifying a first target nucleic acid comprising, providing a sample comprising the first target nucleic acid, providing a first set of paired oligonucleotides with complementarity to the first target nucleic acid, the first set of paired oligonucleotides comprising a first ratio of (a) first competitive oligonucleotides to (b) first signal oligonucleotides comprising a signal tag, wherein the competitive oligonucleotides compete with the signal oligonucleotides for binding to the first target nucleic acid, amplifying the first target nucleic acid with the polymerase chain reaction, thereby degrading the first signal oligonucleotide and permitting generation of a first signal, generating the first signal, measuring intensity of the first signal, and correlating the intensity of the first signal to the first ratio, thereby identifying the first target nucleic acid.

A method of identifying a first target nucleic acid comprising, providing a sample comprising the first target nucleic acid, providing a first set of paired oligonucleotides with complementarity to the first target nucleic acid, the first set of paired oligonucleotides comprising a first ratio of (a) first competitive oligonucleotides to (b) first signal oligonucleotides comprising a signal tag, wherein the competitive oligonucleotides compete with the signal oligonucleotides for binding to the first target nucleic acid, amplifying the first target nucleic acid with the polymerase chain reaction, thereby degrading the first signal oligonucleotide and permitting generation of a first signal, generating the first signal, measuring intensity of the first signal, and correlating the intensity of the first signal to the first ratio, thereby identifying the first target nucleic acid.

Method of analysis for alleles of a target. In the method, a plurality of fluid volumes may be formed. Each fluid volume may contain a first primer pair to amplify a first allele of a target, a second primer pair to amplify a second allele of the target, a first reporter including a first photoluminophore and providing a primer of the first primer pair, and a second reporter including a second photoluminophore and providing a primer of the second primer pair. Each of the first and second reporters may include an oligomer having a quencher, and the oligomer may be configured to base-pair with the primer of the first primer pair and the primer of the second primer pair. The first and second alleles may be amplified using the first and second primer pairs. Photoluminescence may be detected. A level of each allele may be determined.

Method of analysis for alleles of a target. In the method, a plurality of fluid volumes may be formed. Each fluid volume may contain a first primer pair to amplify a first allele of a target, a second primer pair to amplify a second allele of the target, a first reporter including a first photoluminophore and providing a primer of the first primer pair, and a second reporter including a second photoluminophore and providing a primer of the second primer pair. Each of the first and second reporters may include an oligomer having a quencher, and the oligomer may be configured to base-pair with the primer of the first primer pair and the primer of the second primer pair. The first and second alleles may be amplified using the first and second primer pairs. Photoluminescence may be detected. A level of each allele may be determined.

A method of identifying a first target nucleic acid comprising, providing a sample comprising the first target nucleic acid, providing a first set of paired oligonucleotides with complementarity to the first target nucleic acid, the first set of paired oligonucleotides comprising a first ratio of (a) first competitive oligonucleotides to (b) first signal oligonucleotides comprising a signal tag, wherein the competitive oligonucleotides compete with the signal oligonucleotides for binding to the first target nucleic acid, amplifying the first target nucleic acid with the polymerase chain reaction, thereby degrading the first signal oligonucleotide and permitting generation of a first signal, generating the first signal, measuring intensity of the first signal, and correlating the intensity of the first signal to the first ratio, thereby identifying the first target nucleic acid.

A method of identifying a first target nucleic acid comprising, providing a sample comprising the first target nucleic acid, providing a first set of paired oligonucleotides with complementarity to the first target nucleic acid, the first set of paired oligonucleotides comprising a first ratio of (a) first competitive oligonucleotides to (b) first signal oligonucleotides comprising a signal tag, wherein the competitive oligonucleotides compete with the signal oligonucleotides for binding to the first target nucleic acid, amplifying the first target nucleic acid with the polymerase chain reaction, thereby degrading the first signal oligonucleotide and permitting generation of a first signal, generating the first signal, measuring intensity of the first signal, and correlating the intensity of the first signal to the first ratio, thereby identifying the first target nucleic acid.

Certain embodiments of the invention provide a method (i.e., Enzyme Linked Oligonucleotide Sorbent Assay (ELOSA)) for the detection and/or quantification of a test oligonucleotide (e.g., a small oligonucleotide) in a test sample, such as a biological fluid, comprising: a) contacting the test sample with i) a capture reagent bound to a solid support, wherein the capture reagent comprises an oligonucleotide comprising a nucleic acid sequence complementary to the test oligonucleotide; and ii) a competition oligonucleotide operably linked to an enzyme, wherein the competition oligonucleotide comprises a nucleic acid sequence complementary to the capture oligonucleotide; thereby creating a reaction mixture; b) contacting the reaction mixture with a substrate that specifically binds to the enzyme, thereby generating an enzyme-substrate reaction product; and c) measuring the concentration of the enzyme-substrate reaction product, so as to detect and/or quantify the test oligonucleotide.

Certain embodiments of the invention provide a method (i.e., Enzyme Linked Oligonucleotide Sorbent Assay (ELOSA)) for the detection and/or quantification of a test oligonucleotide (e.g., a small oligonucleotide) in a test sample, such as a biological fluid, comprising: a) contacting the test sample with i) a capture reagent bound to a solid support, wherein the capture reagent comprises an oligonucleotide comprising a nucleic acid sequence complementary to the test oligonucleotide; and ii) a competition oligonucleotide operably linked to an enzyme, wherein the competition oligonucleotide comprises a nucleic acid sequence complementary to the capture oligonucleotide; thereby creating a reaction mixture; b) contacting the reaction mixture with a substrate that specifically binds to the enzyme, thereby generating an enzyme-substrate reaction product; and c) measuring the concentration of the enzyme-substrate reaction product, so as to detect and/or quantify the test oligonucleotide.

The invention provides components and methods for polymerase chain reaction assays. The assays minimize both handling of material and time spent running samples. For example, a single internal positive control (IPC) polynucleotide pair can provide a means to ensure proper nucleic acid purification for both RNA and DNA test targets. Additionally, standard cycling conditions for all diagnostic tests allow the user to run both RNA and DNA targets side-by-side.