Enumeration of nucleic acids
Disclosed are methods and systems for enumeration of nucleic acids, including for the detection of rare events in a biological sample. In certain embodiments, the method may comprise arranging polynucleotides obtained from a 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; determining by nucleic acid hybridization (i) a first number of first reaction sites containing a target nucleic acid sequence, or a portion thereof, and (ii) a second number of second reaction sites containing a reference nucleic acid sequence, or a portion thereof; comparing the first number of the first reaction sites to the second number of the second reaction sites to determine the relative amount of the target nucleic acid in the biological sample.
1. A method of determining the relative amount of a target nucleic acid in a biological sample, the method comprising:
amplifying polynucleotides immobilized at the individual sites to generate amplified products,
wherein the polynucleotides are obtained from a biological sample,
wherein the polynucleotides comprise heterogeneous nucleic acids comprising a target nucleic acid and a reference nucleic acid, and
wherein the each individual site contains on average one of the polynucleotides;
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, at respective individual sites on the solid support;
determining (i) a first number of individual sites containing probes hybridized to a target nucleic acid, or a portion thereof, and (ii) a second number of individual sites containing probes hybridized to a reference nucleic acid, or a portion thereof; and
comparing the first number to the second number to determine the relative amount of the target nucleic acid in the biological sample.
2. The method claim 1 , wherein the nucleic acid probes specific for the target nucleic acid, or a portion thereof, are specific for a single locus.
3. The method of claim 1 , wherein the nucleic acid probes specific for the target nucleic acid, or a portion thereof, are specific for multiple loci.
4. The method of claim 1 , wherein the nucleic acid probes specific for the target nucleic acid, or a portion thereof, are multiplexed.
5. The method of claim 4 , wherein the multiplexed probes comprise 2-10,000 probes.
6. The method of claim 5 , wherein the multiplexed probes specific for the target nucleic acid, or a portion thereof, comprise 500 probes.
7. The method of claim 1 , wherein the nucleic acid probes specific for the reference nucleic acid, or a portion thereof, are specific for a single locus.
8. The method of claim 1 , wherein the nucleic acid probes specific for the target nucleic acid, or a portion thereof, are specific for multiple loci.
9. The method of claim 1 , wherein the nucleic acid probes specific for the reference nucleic acid, or a portion thereof, are multiplexed.
10. The method of claim 9 , wherein the multiplexed probes comprise 2-10,000 probes.
11. The method of claim 9 , wherein the multiplexed probes specific for the reference nucleic acid, or a portion thereof, comprise 500 probes.
12. The method claim 1 , wherein the probes specific for the target nucleic acid, or a portion thereof, and the probes specific for the reference nucleic acid, or a portion thereof, are labeled with distinctively detectable signals.
13. The method of claim 12 , wherein the distinctively detectable signals are different optical signals.
14. The method of claim 13 , wherein the different optical signals are distinct fluorescent or luminescent signals.
15. A method for detecting a target nucleic acid in a biological sample, comprising:
amplifying the polynucleotides immobilized at individual sites on a solid support to generate a plurality of clusters such that each cluster contains amplified nucleic acid product
wherein the polynucleotides are obtained from a biological sample and comprise heterogeneous nucleic acids comprising a target nucleic acid and a reference nucleic acid, and wherein the each individual site contains on average one polynucleotide; 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.