Provided herein are methods and systems for sequencing a single nucleic acid molecule utilizing a polymerase enzyme, a template nucleic acid, and a polymerase reagent solution.

Particular aspects provide novel, high-throughput methods to quantify DNA methylation (e.g., at a single-base resolution) in an allele-specific manner. The methods comprise use of an allele-specific sequence polymorphism (e.g., allele-specific single nucleotide polymorphism; SNP) in sufficient proximity to a CpG methylation site to provide for distinguishing the methylation levels between two alleles. In particular aspects, after bisulfite modification, the genomic DNA region is PCR-amplified, and the product subjected to allele-specific pyrosequencing, and the percentage of methylation determined based on the percentage of cytosine to thymidine conversion. In further embodiments, MethyLight™ is used after bisulfite treatment. The inventive methodology has, for example, substantial utility for affording quantitative analyses in the regulation of analyses of X-inactivation, the allele-specific expression of genes (e.g., in the immune system) and junk DNA, etc., and in classifying an individual as to whether they have loss of imprinting (LOI).

Particular aspects provide novel, high-throughput methods to quantify DNA methylation (e.g., at a single-base resolution) in an allele-specific manner. The methods comprise use of an allele-specific sequence polymorphism (e.g., allele-specific single nucleotide polymorphism; SNP) in sufficient proximity to a CpG methylation site to provide for distinguishing the methylation levels between two alleles. In particular aspects, after bisulfite modification, the genomic DNA region is PCR-amplified, and the product subjected to allele-specific pyrosequencing, and the percentage of methylation determined based on the percentage of cytosine to thymidine conversion. In further embodiments, MethyLight™ is used after bisulfite treatment. The inventive methodology has, for example, substantial utility for affording quantitative analyses in the regulation of analyses of X-inactivation, the allele-specific expression of genes (e.g., in the immune system) and junk DNA, etc., and in classifying an individual as to whether they have loss of imprinting (LOI).

The invention provides for sequencing a nucleic acid molecule based on the detection of base incorporation by the release of pyrophosphate (PPi) using a new enzyme system comprising adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) and its substrate ADP-glucose.

The invention provides for sequencing a nucleic acid molecule based on the detection of base incorporation by the release of pyrophosphate (PPi) using a new enzyme system comprising adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) and its substrate ADP-glucose.

An assembly and a method are disclosed for analyzing nucleic acid sequences by way of so-called sequencing-by-synthesis. According to an embodiment of the invention, a chemical substance group that is released when a nucleotide bonds to a nucleic acid sequence to be sequenced is detected. The reagents are applied by way of a spraying device to a sensor that detects the released substance group. This has the advantage that no lateral flow occurs. The rate of false-negative and false-positive results is significantly reduced. Furthermore, a small amount of the reagent is sufficient to completely wet the sensor. Filling of the supply and discharge lines as for a flow cell is not necessary.

An assembly and a method are disclosed for analyzing nucleic acid sequences by way of so-called sequencing-by-synthesis. According to an embodiment of the invention, a chemical substance group that is released when a nucleotide bonds to a nucleic acid sequence to be sequenced is detected. The reagents are applied by way of a spraying device to a sensor that detects the released substance group. This has the advantage that no lateral flow occurs. The rate of false-negative and false-positive results is significantly reduced. Furthermore, a small amount of the reagent is sufficient to completely wet the sensor. Filling of the supply and discharge lines as for a flow cell is not necessary.

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

Provided herein are methods and systems for sequencing a single nucleic acid molecule utilizing a polymerase enzyme, a template nucleic acid, and a polymerase reagent solution.