Methods and compositions for assessing nucleic acids
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 of assessing a first nucleic acid in a first sample comprising:
providing a first sample comprising a first nucleic acid;
amplifying said first nucleic acid; and
obtaining a relationship wherein said relationship can enumerate less than about 1,000 molecules of said first nucleic acid in said first sample.
2 . The method as recited in claim 1 wherein said relationship can enumerate less than about 100 molecules of said first nucleic acid in said first sample.
3 . The method as recited in claim 1 wherein said relationship compares a first relationship of amplified product of said first nucleic acid to co-amplified product of a competitive template for said first nucleic acid to a second relationship of amplified product of a second nucleic acid in said first sample to co-amplified product of a competitive template for said second nucleic acid.
4 . The method as recited in claim 3 wherein said competitive template for said first or said second nucleic acid comprises a sequence referenced in Table 4.
5 . The method as recited in claim 3 wherein said second nucleic acid serves as a first reference nucleic acid.
6 . The method as recited in claim 3 wherein said first reference nucleic acid is a control for loading.
7 . The method as recited in claim 6 wherein said first reference nucleic acid corresponds to at least one gene selected from GADP, ACTB, and β-actin.
8 . The method as recited in claim 3 wherein said relationship further compares amplified product of a number of other nucleic acid(s) to co-amplified product of competitive template(s) for said number of other nucleic acid(s).
9 . The method as recited in claim 1 wherein obtaining said relationship comprises a use of microfluidic capillary electrophoresis, an oligonucleotide array, mass spectrometry, or chromatography.
10 . The method as recited in claim 1 wherein obtaining said relationship does not involve taking real-time measurements nor generation of a standard curve.
11 . The method as recited in claim 10 wherein said relationship controls for at least two sources of variation selected from cDNA loading, intra-nucleic acid amplification efficiency, inter-nucleic acid amplification efficiency, inter-specimen amplification efficiency, inter-sample amplification efficiency, and intra-sample amplification efficiency.
12 . The method as recited in claim 1 wherein said relationship is capable of detecting less than about a two-fold difference.
13 . The method as recited in claim 1 wherein said relationship is capable of detecting less than about an 80% difference.
14 . The method as recited in claim 1 wherein said relationship is capable of detecting less than about a two-fold difference in about 100 molecules or less of said first nucleic acid in said first sample.
15 . The method as recited in claim 14 wherein said relationship is capable of detecting less than about an 80% difference.
16 . The method as recited in claim 1 wherein said relationship is capable of detecting less than about a two-fold difference in about 10 molecules or less of said first nucleic acid in said first sample.
17 . The method as recited in claim 16 wherein said relationship is capable of detecting less than about an 80% difference.
18 . The method as recited in claim 1 wherein said relationship is capable of detecting less than about a two-fold difference in about 1 molecule or less of said first nucleic acid in said first sample.
19 . The method as recited in claim 18 wherein said relationship is capable of detecting less than about an 80% difference.
20 . The method as recited in claim 1 wherein said relationship provides a coefficient of variation of less than about 25% between said first sample and a second sample of said first nucleic acid.
21 . The method as recited in claim 1 wherein said relationship provides a coefficient of variation of less than about 5% between said first sample and a second sample of said first nucleic acid.
22 . The method as recited in claim 20 or 21 wherein said first and said second samples are amplified at different times.
23 . The method as recited in claim 1 wherein said first nucleic acid comprises a sequence referenced in Table 1 or 2.
24 . The method as recited in claim 1 wherein said relationship reduces false negatives.
25 . The method as recited in claim 1 wherein said nucleic acid comprises an RNA molecule.
26 . The method as recited in claim 1 wherein said nucleic acid comprises a DNA molecule.
27 . The method as recited in claim 1 wherein said relationship is substantially constant beyond an exponential phase of said amplification of said first nucleic acid.
28 . The method as in claim 1 wherein said amplifying of said first nucleic acid comprises:
co-amplifying said first nucleic acid, a number of other nucleic acid(s), a competitive template for said first nucleic acid and a competitive template(s) for said other nucleic acid(s) wherein said competitive templates are at known concentrations relative to one another, to produce first amplified product thereof;
diluting said first amplified product; and
further co-amplifying said diluted first amplified product of said first nucleic acid and of said competitive template for said first nucleic acid, to produce second amplified product thereof.
29 . The method as recited in claim 28 wherein said number is one other nucleic acid.
30 . The method as recited in claim 28 wherein said diluting produces at least about a 100-fold dilution.
31 . The method as recited in claim 28 wherein one of said other nucleic acids serves as a first reference nucleic acid.
32 . The method as recited in claim 31 wherein said assessing comprises:
obtaining a first relationship, said first relationship comparing said second amplified product of said first nucleic acid to said second amplified product of said competitive template for said first nucleic acid;
obtaining a second relationship, said second relationship comparing said first amplified product of said first reference nucleic acid to said first amplified product of said competitive template for said first reference nucleic acid; and
comparing said first and said second relationships.
33 . The method as recited in claim 31 wherein another one of said other nucleic acids serves as a second reference nucleic acid.
34 . The method as recited in claim 33 wherein said assessing comprises:
obtaining a first relationship, said first relationship comparing said second amplified product of said first nucleic acid to said second amplified product of said competitive template for said first nucleic acid;
obtaining a third relationship, said third relationship comparing said first amplified product of said second reference nucleic acid to said first amplified product of said competitive template for said second reference nucleic acid; and
comparing said first and said third relationships.
35 . The method as recited in claim 31 , further comprising diluting and further co-amplifying said diluted first amplified product of said first reference nucleic acid and of said competitive template for said first reference nucleic acid, to produce second amplified products thereof.
36 . The method as recited in claim 35 wherein said assessing comprises:
obtaining a first relationship, said first relationship comparing said second amplified product of said first nucleic acid to said second amplified product of said competitive template for said first nucleic acid;
obtaining a fourth relationship, said fourth relationship comparing said second amplified product of said first reference nucleic acid to said second amplified product of said competitive template for said first reference nucleic acid; and
comparing said first and said fourth relationships.