The present disclosure generally relates to artificial controls for genetic sequencing and quantitation assays, which can be used to calibrate a wide variety of genetic sequencing and quantitation methods. For example, the controls disclosed herein can be used to calibrate a wide variety of high throughput sequencing methods (for example, those referred to as next generation sequencing methods). The present disclosure also generally relates to the use of the sequencing controls in a wide variety of applications including, for example, in the calibration of a wide variety of sequencing methods.

Methods for obtaining a reaction volume having a predetermined copy number, such as a single copy, of a known nucleic acid molecule therein are disclosed. Such a reaction volume may be useful as a control for nucleic acid amplification reactions. Nucleic acid constructs useful in such methods are also described. The constructs include a first region and a second region, wherein the second region is flanked by first and second primer binding sites to allow amplification across the second region, and wherein a selectively cleavable region is located between the first and second regions, with the selectively cleavable region being flanked by third and fourth primer binding sites to allow amplification across the selectively cleavable region. The second region acts as a reporter to indicate the presence of the entire nucleic acid molecule; and the selectively cleavable region can be cleaved to separate the first region from the second region, leaving an isolated copy of the first region.

Methods for obtaining a reaction volume having a predetermined copy number, such as a single copy, of a known nucleic acid molecule therein are disclosed. Such a reaction volume may be useful as a control for nucleic acid amplification reactions. Nucleic acid constructs useful in such methods are also described. The constructs include a first region and a second region, wherein the second region is flanked by first and second primer binding sites to allow amplification across the second region, and wherein a selectively cleavable region is located between the first and second regions, with the selectively cleavable region being flanked by third and fourth primer binding sites to allow amplification across the selectively cleavable region. The second region acts as a reporter to indicate the presence of the entire nucleic acid molecule; and the selectively cleavable region can be cleaved to separate the first region from the second region, leaving an isolated copy of the first region.

Methods and compositions for detecting genetic instability using digital amplification assays. The methods may be performed in a set of isolated volumes and generally may involve competitive hybridization of a competitor and a probe/primer with a normal allele and one or more mutant alleles of a microsatellite locus. The competitor may be configured to compete similarly with, or to outcompete, the primer/probe for hybridization with the normal allele. The primer/probe may be configured to outcompete the competitor for hybridization with various mutant alleles of the locus that alter the length of the repetitive sequence by different amounts. Isolated volumes in which the primer/probe outcompetes the competitor may be enumerated, and represent one or more of the mutant alleles. The methods may enable diagnosing microsatellite instability and treating a subject based on the diagnosis.

Methods and compositions for detecting genetic instability using digital amplification assays. The methods may be performed in a set of isolated volumes and generally may involve competitive hybridization of a competitor and a probe/primer with a normal allele and one or more mutant alleles of a microsatellite locus. The competitor may be configured to compete similarly with, or to outcompete, the primer/probe for hybridization with the normal allele. The primer/probe may be configured to outcompete the competitor for hybridization with various mutant alleles of the locus that alter the length of the repetitive sequence by different amounts. Isolated volumes in which the primer/probe outcompetes the competitor may be enumerated, and represent one or more of the mutant alleles. The methods may enable diagnosing microsatellite instability and treating a subject based on the diagnosis.

System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

The invention relates to a method for amplification of a nucleic acid by a nucleic acid polymerase via two primers, each of which comprise a sequence segment which is bound to the target sequence and a sequence segment which does not bind to the target sequence, wherein the second cannot serve as a template for the polymerase, and two controller oligonucleotides, each of which is complementary to the primers and a segment of the sequence segment synthesized by them and serves to release the synthesized strand from the template. The controllers also comprise modified nucleotide building blocks such that they cannot serve as templates for the activity of the first template-dependent nucleic acid polymerase.

The invention relates to a method for amplification of a nucleic acid by a nucleic acid polymerase via two primers, each of which comprise a sequence segment which is bound to the target sequence and a sequence segment which does not bind to the target sequence, wherein the second cannot serve as a template for the polymerase, and two controller oligonucleotides, each of which is complementary to the primers and a segment of the sequence segment synthesized by them and serves to release the synthesized strand from the template. The controllers also comprise modified nucleotide building blocks such that they cannot serve as templates for the activity of the first template-dependent nucleic acid polymerase.

The present invention relates to a method for the extension of an oligonucleotide primer with improved specificity using a specific oligonucleotide primer and a controller nucleotide, wherein the controller oligonucleotide enables sequence-specific strand opening of a double strand of extension product and template. The invention further relates to a respective kit for carrying out the method according to the invention.