Methods and compositions for assessing nucleic acids and alleles
The present invention is directed to methods and compositions for evaluating allelic variations with improved quality controls, methods of preparing such compositions, and applications employing such compositions and methods. In particular, the present invention provides methods and compositions for reducing false positives and/or false negatives in nucleic acid measurements.
1. A method of assessing allele frequency of a first allele and a second allele of a target nucleic acid in a sample, comprising:
co-amplifying said target nucleic acid and a known amount of a competitive template for said target nucleic acid;
co-amplifying a reference nucleic acid and a known amount of a competitive template for said reference nucleic acid;
obtaining a first relation, said first relation comparing amplified product of said target nucleic acid corresponding to said first allele to amplified product of said competitive template for said target nucleic acid;
obtaining a second relation, said second relation comparing amplified product of said target nucleic acid corresponding to said second allele to amplified product of said competitive template for said target nucleic acid;
obtaining a third relation, said third relation comparing amplified product of said reference nucleic acid to amplified product of said competitive template for said reference nucleic acid;
comparing one of said first, second or third relations to both of said other relations, thereby assessing allele frequency of said first and said second alleles in said sample; and
detecting false positives of said first allele by the steps of: co-amplifying a known amount of positive control for said second allele and a known amount of said competitive template for said target nucleic acid; and obtaining a fourth relation, said fourth relation comparing amplified product of said positive control for said second allele that is detectable by a detection moiety specific for said first allele to amplified product of said competitive template for said target nucleic acid,
wherein obtaining said first, second and/or third relation comprises a use of an oligonucleotide array and wherein said amplified products of said target nucleic acid
corresponding to said first allele, of said target nucleic acid corresponding to said second allele and of said competitive template for said target nucleic acid are immobilized by a first common capture moiety.
2. A method of assessing allele frequency of a first allele and a second allele of a target nucleic acid in a sample, comprising:
co-amplifying said target nucleic acid and a known amount of a competitive template fir said target nucleic acid;
co-amplifying a reference nucleic acid and a known amount of a competitive template for said reference nucleic acid;
obtaining a first relation, said first relation comparing amplified product of said target nucleic acid corresponding to said first allele to amplified product of said competitive template for said target nucleic acid;
obtaining a second relation, said second relation comparing amplified product of said target nucleic acid corresponding to said second allele to amplified product of said competitive template for said target nucleic acid;
obtaining a third relation, said third relation comparing amplified product of said reference nucleic acid to amplified product of said competitive template for said reference nucleic acid;
comparing one of said first, second or third relations to both of said other relations, thereby assessing allele frequency of said first and said second alleles in said sample; and
detecting false positives of said first allele by the steps of: co-amplifying a known amount of positive control for said second allele and a known amount of said competitive template for said target nucleic acid; and obtaining a fourth relation, said fourth relation comparing amplified product of said positive control for said second allele that is detectable by a detection moiety specific for said first allele to amplified product of said competitive template for said target nucleic acid,
wherein obtaining said first, second and/or third relation comprises a use of an oligonucleotide array and wherein said amplified products of said reference nucleic acid and of said competitive template for said reference nucleic acid are immobilized by a second common capture moiety.
3. The method as recited in claim 1 or 2 , wherein said target nucleic acid comprises genomic DNA.
4. The method as recited in claim 1 or 2 , wherein said target nucleic acid comprises mRNA.
5. The method as recited in claim 1 or 2 , wherein said target nucleic acid comprises cDNA.
6. The method as recited in claim 1 or 2 , wherein said sample comprises pooled nucleic acid from different subjects.
7. The method as recited in claim 1 or 2 , wherein said sample comprises nucleic acid from a disease state.
8. The method as recited in claim 1 or 2 , wherein said first and said second alleles comprise single nucleotide polymorphisms.
9. The method as recited in claim 1 or 2 , wherein said method does not involve real time measurements.
10. The method as recited in claim 1 or 2 , wherein said method does not involve generation of a standard curve.
11. The method as recited in claim 1 or 2 , wherein said method does not use two-color labeling.
12. The method as recited in claim 1 or 2 , wherein said method does not use allele-specific primers.
13. The method as recited in claim 1 or 2 , wherein said method does not use a universal priming sequence.
14. The method as recited in claim 1 or 2 , wherein method does not detect aneuploidy.
15. The method as recited in claim 1 or 2 , wherein said method does not use pyrophosphorolysis.
16. The method as recited in claim 1 or 2 , wherein said method does not use gel electrophoresis.
17. The method as recited in claim 1 or 2 , wherein obtaining said first, second and/or third relation comprises immobilization of said amplified products.
18. The method as recited in claim 17 , wherein at least two of said immobilized amplified products are distinguished using at least two detection moieties selected from a detection moiety specific for said first allele, a detection moiety specific for said second allele, and a detection moiety specific for said competitive template for said target nucleic acid.
19. The method as recited in claim 18 , wherein said immobilized amplified products are distinguished using a detection moiety specific for said reference nucleic acid and a detection moiety specific for said competitive template for said reference nucleic acid.
20. The method as recited in claim 1 or 2 , further comprising detecting false negatives for said first allele by the steps of: co-amplifying a known amount of a positive control for said first allele and a known amount of said competitive template for said target nucleic acid; and obtaining a fifth relation, said fifth relation comparing amplified product of said positive control for said first allele that is detectable by a detection moiety specific for said first allele to amplified product of said competitive template for said target nucleic acid.
21. The method as recited in claim 20 , wherein said positive control for said first allele is co-amplified with a substantially equal amount of said competitive template for said target nucleic acid.
22. The method as recited in claim 20 , wherein said false negatives are reduced.
23. The method as recited in claim 20 , wherein said false negatives are eliminated.
24. The method as recited in claim 1 or 2 , wherein said positive control for said second allele is co-amplified with a substantially equal amount of said competitive template for said target nucleic acid.
25. The method as recited in claim 1 or 2 , wherein said false positives are reduced.
26. The method as recited in claim 1 or 2 , further comprising detecting false negatives of said second allele by the steps of: co-amplifying a known amount of a positive control for said second allele and a known amount of said competitive template for said target nucleic acid; and obtaining a sixth relation, said sixth relation comparing amplified product of said positive control for said second allele that is detectable by a detection moiety specific for said second allele to amplified product of said competitive template for said target nucleic acid.
27. The method as recited in claim 26 , wherein said positive control for said second allele is co-amplified with a substantially equal amount of said competitive template for said target nucleic acid.
28. The method as recited in claim 26 , wherein said false negatives are reduced.
29. The method as recited in claim 26 , wherein said false negatives are eliminated.
30. The method as recited in claim 1 or 2 , further comprising detecting false positives of said second allele by the steps of: co-amplifying a known amount of positive control for said first allele and a known amount of said competitive template for said target nucleic acid; and obtaining a seventh relation, said seventh relation comparing amplified product of said positive control for said first allele that is detectable by a detection moiety specific for said second allele to amplified product of said competitive template for said target nucleic acid.
31. The method as recited in claim 30 , wherein said positive control for said first allele is co-amplified with a substantially equal amount of said competitive template for said target nucleic acid.
32. The method as recited in claim 30 , wherein said false positives are reduced.
33. The method as recited in claim 1 or 2 , wherein said first and said second relation are substantially constant beyond an exponential phase of said amplification of said target nucleic acid.
34. The method as recited in claim 1 or 2 , wherein the co-amplifying of said target nucleic acid and said competitive template for said target nucleic acid employs some primers, the method further comprising, the method further comprising: diluting said co-amplified products of said target nucleic acid and of said competitive template for said target nucleic acid; and further co-amplifying said diluted amplified products using the same primers as used in the initial co-amplifying step.
35. The method as recited in claim 1 or 2 , wherein less than about 100 molecules of said target nucleic acid in said sample are amplified.
36. The method as recited in claim 1 or 2 , wherein said competitive templates are provided in a standardized mixture at known concentrations relative to each other.
37. The method as recited in claim 1 or 2 , wherein said competitive template for said target nucleic acid is provided at a series of dilutions relative to said competitive template for said reference nucleic acid.
38. The method as recited in claim 1 or 2 , wherein said method is computer implemented.
39. The method as recited in claim 2 , wherein said immobilized amplified products are distinguished using a detection moiety specific for said reference nucleic acid and a detection moiety specific for said competitive template for said reference nucleic acid.