Thermal reaction device and method for using the same
View Patent ↗Devices and methods for performing the relative concentration of a target in a sample, the sample containing both target and non-target components, the method performed by partitioning the sample into a large number of reaction volumes such that the target is concentrated relative to the non-target, and performing a detection assay upon each reaction volume to detect the target.
1. A method for highly sensitive and specific detection of mutant alleles in a high background of wild-type alleles, comprising
a) obtaining an assay composition comprising first polynucleotides encoding a mutant allele of a gene and second polynucleotides encoding a wild-type allele of the gene, wherein the ratio of first polynucleotides to second polynucleotides is 1:1×10 3 or lower;
b) distributing the assay composition, or a portion thereof, into at least 10,000 aliquots, thereby producing a subset of aliquots in which the ratio of first polynucleotides to the second polynucleotides is greater than the ratio of first polynucleotides to second polynucleotides in said assay composition, wherein some aliquots comprise a first polynucleotide and essentially all aliquots comprise at least one second polynucleotide;
c) combining the assay composition with amplification reagents prior to step (b) or combining the at least 10,000 aliquots with amplification reagents after step (b), wherein said amplification reagents:
i) comprise amplification primers specific for amplifying at least a portion of the mutant allele, and
ii) do not comprise amplification primers specific for amplifying the wild-type allele;
d) amplifying the at least a portion of the mutant allele encoded in the first polynucleotides; and
e) detecting the presence or absence of amplification in each aliquot.
2. The method of claim 1 wherein the first and second polynucleotides are derived from the genome of the same individual.
3. The method of claim 1 wherein said assay composition is combined with all of said amplification reagents prior to step (b).
4. The method of claim 1 wherein the mutant allele differs from the wild-type allele by one or more point mutations.
5. The method of claim 1 wherein the mutant allele differs from the wild-type allele by an insertion or deletion.
6. The method of claim 1 wherein the amplification in (d) is by real-time PCR.
7. The method of claim 1 wherein the amplification mixture additionally comprises a nonextendible primer that binds specifically to the wild-type polynucleotide.
8. The method of claim 1 wherein the ratio of first polynucleotides to second polynucleotides is 1:1×10 5 or lower.
9. The method of claim 1 , wherein the presence of amplification is determined by detecting a fluorescent signal.
10. The method of claim 1 wherein the gene is an oncogene.
11. The method of claim 1 wherein the assay composition is combined with amplification reagents prior to step (b).
12. The method of claim 1 wherein some aliquots comprise a first polynucleotide and essentially all aliquots comprise at least a plurality of second polynucleotides.
13. The method of claim 12 wherein some aliquots comprise a first polynucleotide and essentially all aliquots comprise hundreds of second polynucleotides.