Enumeration Of Nucleic Acids

Abstract

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:
 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.   
     
     
         2 . 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.   
     
     
         3 . The method of  claim 1 , wherein the method further comprises a step of harvesting the amplified products before the hybridizing step. 
     
     
         4 . The method of  claim 3 , wherein the hybridizing step is carried out with a microarray. 
     
     
         5 . The method of  claim 1 , wherein the arranging step is performed using a solid, semi-solid and/or liquid support. 
     
     
         6 . The method of  claim 5 , wherein the arranging step is performed on a solid support. 
     
     
         7 . The method of  claim 6 , wherein the solid support is selected from the group consisting of glasses, optical fiber, silica, microchips, plastics, beads; biofilms, cellulose. 
     
     
         8 . The method of  claim 6 , wherein the arranging step comprises a step of using flowcell technology. 
     
     
         9 . The method of  claim 6 , wherein the arranging step comprises a step of distributing the collection of nucleic acid molecules within a gel matrix. 
     
     
         10 . The method of  claim 6 , wherein the arranging step comprises a step of mixing the collection of nucleic acid molecules with emulsion beads. 
     
     
         11 . The method of  claim 5  wherein the support comprises a plurality of capturing moieties immobilized thereon to capture individual polynucleotide. 
     
     
         12 . The method of  claim 11 , wherein the capturing moieties comprise capturing oligonucleotides. 
     
     
         13 . The method of  claim 12 , wherein the method further comprises a step of first treating the polynucleotides such that individual polynucleotides contain adapter sequences at the 5′ and 3′ ends, wherein the adapter sequences hybridize to the capturing oligonucleotides immobilized. 
     
     
         14 . The method of  claim 1 , wherein the amplifying step comprises performing a PCR reaction. 
     
     
         15 . The method of  claim 14 , wherein the amplifying step comprises performing rolling circle amplification. 
     
     
         16 . The method of  claim 1 , wherein the target nucleic acid is a target chromosome. 
     
     
         17 . The method of  claim 16 , wherein the target chromosome is selected from chromosome X, Y, 13, 18, or 21. 
     
     
         18 . The method of  claim 1 , wherein the reference nucleic acid is a reference chromosome. 
     
     
         19 . The method of  claim 18 , wherein the reference chromosome is chromosome 1, X, Y, 13, 18, or 21. 
     
     
         20 . The method of  claim 1 , wherein the target nucleic acid contains a mutation. 
     
     
         21 . The method of  claim 20 , wherein the mutation is selected from the group consisting of SNPs, deletions, insertions, duplications, and combinations thereof. 
     
     
         22 . The method of  claim 1 , wherein the biological sample comprises over-abundance or under-abundance of the target nucleic acid. 
     
     
         23 . The method of  claim 22 , wherein the biological sample comprises aneuploid chromosomes. 
     
     
         24 . The method  claim 2 , wherein the nucleic acid probes specific for the target nucleic acid, or a portion thereof, are specific for a single locus. 
     
     
         25 . The method of  claim 2 , wherein the nucleic acid probes specific for the target nucleic acid, or a portion thereof, are specific for multiple loci. 
     
     
         26 . The method of  claim 2 , wherein the nucleic acid probes specific for the target nucleic acid, or a portion thereof, are multiplexed. 
     
     
         27 . The method of  claim 26 , wherein the multiplexed probes comprise 2-10,000 probes. 
     
     
         28 . The method of  claim 27 , wherein the multiplexed probes specific for the target nucleic acid, or a portion thereof, comprise 500 probes. 
     
     
         29 . The method of  claim 2 , wherein the nucleic acid probes specific for the reference nucleic acid, or a portion thereof, are specific for a single locus. 
     
     
         30 . The method of  claim 2 , wherein the nucleic acid probes specific for the target nucleic acid, or a portion thereof, are specific for multiple loci. 
     
     
         31 . The method of  claim 2 , wherein the nucleic acid probes specific for the reference nucleic acid, or a portion thereof, are multiplexed. 
     
     
         32 . The method of  claim 31 , wherein the multiplexed probes comprise 2-10,000 probes. 
     
     
         33 . The method of  claim 31 , wherein the multiplexed probes specific for the reference nucleic acid, or a portion thereof, comprise 500 probes. 
     
     
         34 . The method  claim 2 , 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. 
     
     
         35 . The method of  claim 34 , wherein the distinctively detectable signals are different optical signals. 
     
     
         36 . The method of  claim 35 , wherein the different optical signals are distinct fluorescent or luminescent signals. 
     
     
         37 . The method  claim 1 , wherein the determining step comprises determining the relative copy number of the target nucleic acid. 
     
     
         38 . The method of  claim 37 , wherein the step of determining the relative copy number of the target nucleic acid comprises the steps of
 (a) determining a first fluorescent signal level indicative of the first number of the detection sites containing the target nucleic acid, or a portion thereof, normalized to the background signals;   (b) determining a second fluorescent signal level indicative of the second number of the detection sites containing the reference nucleic acid, or a portion thereof, normalized to the background signals;   (c) determining a ratio of the first fluorescent signal level to the second fluorescent signal level; and   (d) determining the relative copy number of the target nucleic acid by normalizing the ratio determined at step (c) to the ratio between the number of probes specific for the target nucleic acid and the number of probes specific for the reference nucleic acid.   
     
     
         39 . The method of  claim 1 , wherein the polynucleotides obtained from the biological sample contain genomic DNA in an amount less than one genome equivalence. 
     
     
         40 . The method of  claim 1 , wherein the amplified products contain amplified genomic DNA at a pre-determined level of genome equivalence. 
     
     
         41 . The method  claim 1 , wherein less than 1% of the polynucleotides obtained from the biological sample represent the target nucleic acid. 
     
     
         42 . The method of  claim 1 , wherein less than 0.1% of the polynucleotides obtained from the biological sample represent the target nucleic acid. 
     
     
         43 . The method of  claim 1 , wherein less than 1 out of a million of the polynucleotides obtained from the biological sample represent the target nucleic acid. 
     
     
         44 . The method of  claim 1 , wherein less than 1 out of 10 million of the polynucleotides obtained from the biological sample represent the target nucleic acid. 
     
     
         45 . The method of  claim 1 , wherein the target nucleic acid is derived from a fetal cell. 
     
     
         46 . The method of  claim 45 , wherein the target nucleic acid comprises a fetal chromosome. 
     
     
         47 . The method of  claim 1 , wherein the target nucleic acid is derived from a diseased cell. 
     
     
         48 . The method of  claim 47 , wherein the diseased cell is a cancer cell. 
     
     
         49 . The method of  claim 1 , wherein the method further comprises a step of whole genome amplification prior to the arranging step. 
     
     
         50 . The method of  claim 1 , wherein the method further comprises a step of determining if the relative amount of the target nucleic acid is abnormal as compared to a control. 
     
     
         51 . The method of  claim 1 , wherein the method is used to detect a disease, disorder or condition, or a carrier thereof, associated with the abnormal amount of the target nucleic acid. 
     
     
         52 . The method of  claim 1 , wherein the method is used for prenatal diagnosis of fetal aneuploidy. 
     
     
         53 . The method of  claim 1 , wherein the biological sample is selected from the group consisting of cells, tissue, whole blood, plasma, serum, urine, stool, saliva, cord blood, chorionic villus sample, chorionic villus sample culture, amniotic fluid, amniotic fluid culture, transcervical lavage fluid, and combination thereof. 
     
     
         54 . 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.   
     
     
         55 . 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.