Enumeration of Nucleic Acids

A method of determining the relative amount of a target nucleic acid in a biological sample, the method comprising:

• arranging polynucleotides obtained from a biological sample to form a plurality of amplification sites, wherein each amplification site contains on average one polynucleotide;
• amplifying polynucleotides in the plurality of amplification sites to generate amplified products;
• hybridizing the amplified products with nucleic acid probes specific for (i) a target nucleic acid, or a portion thereof, and (ii) a reference nucleic acid, or a portion thereof, in a plurality of detection sites, wherein each detection site contains on average one polynucleotide from the amplified products;
• determining (i) a first number of first detection sites containing probes hybridized to a target nucleic acid, or a portion thereof, and (ii) a second number of second detection sites containing probes hybridized to a reference nucleic acid, or a portion thereof;
• and
• comparing the first number of the first detection sites to the second number of the second detection sites to determine the relative amount of the target nucleic acid in the biological sample.

A method for detecting a target nucleic acid in a biological sample, comprising:

• arranging polynucleotides obtained from the biological sample to form a plurality of reaction sites, wherein each reaction site contains on average one polynucleotide;
• amplifying the polynucleotides in the plurality of reaction sites to generate a plurality of clusters such that each cluster contains amplified nucleic acid product;
• and
• detecting clusters or reaction sites containing the target nucleic acid, or a portion thereof, by hybridization using one or more probes specific for the target nucleic acid, thereby detecting the target nucleic acid in the biological sample.

A system for determining the relative amount of a target nucleic acid, comprising:

• means to amplify polynucleotides on a plurality of amplification sites to generate amplified products;
• means to carry out hybridization of the amplified products with probes labeled with first detectable signals that are specific for a target nucleic acid, or a portion thereof, and probes labeled with second detectable signals that are specific for a reference nucleic acid, or a portion thereof;
• a determination module adapted to (i) determine the level of the first detectable signals, and (ii) determine the level of the second detectable signals;
• a storage device configured to store (iii) signal information from the determination module and (iv) the information provided by a user indicative of the number of the probes specific for the target nucleic acid, or a portion thereof, and the number of the probes specific for the reference nucleic acid, or a portion thereof;
• and
• a computing module adapted to (v) calculate the ratio of the level of the first detectable signals to the level of the second detectable signals and (vi) determine the relative amount of the target nucleic acid by normalizing the ratio of the signal levels to the ratio between the number of the probes specific for the target nucleic acid, or a portion thereof, and the number of the probes specific for the reference nucleic acid, or a portion thereof.