Compositions of standardized mixtures for determining an amount of a nucleic acid
The present invention is directed to methods and compositions for evaluating nucleic acids, methods of preparing such compositions, and applications and business methods employing such compositions and methods. In particular, the present invention provides business methods for operating a gene expression measurement service.
1. A method for preparing a standardized mixture for assessing amounts of at least one target nucleic acid in a sample, the method comprising
providing a first competitive template for a first target nucleic acid in the sample, wherein the first competitive template comprises a first competitive template nucleic acid at a first concentration,
providing a second competitive template for a second target nucleic acid in the sample, wherein the second competitive template comprises a second competitive template nucleic acid at a second concentration that is different from the first concentration, and
combining said first concentration of said first competitive template and said second concentration of said second competitive template in the sample, wherein said first and second competitive templates are at known different concentrations relative to each other, to form said standardized mixture for assessing amounts of at least one target nucleic acid and for allowing direct comparison of said amounts between said first and second nucleic acids in the sample,
wherein a competitive template for a target nucleic acid comprises a competitive template nucleic acid that can be amplified using identical primers, shares sequence homology, and amplifies with substantially similar efficiency as the target nucleic acid while having one or more features that allow the competitive template to be distinguished from the target nucleic acid selected from the group consisting of: one or more point mutations; one or more insert mutations, one or more inversion mutations; and one or more deletion mutations.
2. The method as recited in claim 1 wherein said method further comprise using a forward primer and/or a reverse primer for priming amplification of at least one of said first and second competitive templates.
3. A method for preparing a series of serially-diluted standardized mixtures of reagents for assessing amounts of at least one target nucleic acid in a sample, the method comprising:
providing a first competitive template for a first target nucleic acid in the sample, wherein the first competitive template comprises a first competitive template nucleic acid at a first series of dilution concentrations, and
a second competitive template for a second target nucleic acid in the sample, wherein the second competitive template comprises a second competitive template nucleic acid at a second series of dilution concentrations that are different from the first series of dilution concentrations in a first sample, wherein said first and second competitive templates are at known different and serially diluted concentrations relative to each other, and
combining said first series of concentrations of said first competitive template and said second series of concentrations of said second competitive template to form said standardized mixtures for assessing amounts at least one nucleic acid and for allowing direct comparison of said amounts between said first and second nucleic acids in the sample,
wherein a competitive template for a target nucleic acid comprises a competitive template nucleic acid that can be amplified using identical primers, shares sequence homology, and amplifies with substantially similar efficiency as the target nucleic acid while having one or more features that allow the competitive template to be distinguished from the target nucleic acid selected from the group consisting of: one or more point mutations; one or more insert mutations, one or more inversion mutations; and one or more deletion mutations.
4. The method of claim 1 , wherein a ratio of said first competitive template nucleic acid to said second competitive template is greater than, or about, 1/10.
5. The method of claim 3 , wherein said serial diluted concentrations of said first competitive template, relative to a concentration of said second competitive template comprises one or more of:
mixture A=10 6 molecules/μL;
mixture B=10 5 molecules/μL;
mixture C=10 4 molecules/μL;
mixture D=10 3 molecules/μL;
mixture E=10 2 molecules/μL; and
mixture F=10 1 molecules/μL.
6. The method of claim 5 , wherein said serial diluted concentrations comprise mixtures A, B, C, D, E and F.
7. The method of claim 3 , wherein said serial diluted concentrations of said first competitive template, relative to said concentration of said second competitive template comprises one or more of:
mixture A=10 6 molecules/μL;
mixture B=10 5 molecules/μL;
mixture C=10 4 molecules/μL;
mixture D=10 3 molecules/μL;
mixture E=10 2 molecules/μL; and
mixture F=10 1 molecules/μL.
8. The method of claim 7 , wherein said serial diluted concentrations comprise mixtures A, B, C, D, E and F.
9. The method of claim 1 , wherein a ratio of said first competitive template nucleic acid to said second competitive template nucleic acid is less than, or about, 10/1.