Method for quantification of an analyte

• US 6,303,305 B1
• Filed: 03/30/1999
• Issued: 10/16/2001
• Est. Priority Date: 03/30/1999
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG), University of Utah Research Foundation (State of Utah)
• Original Assignee: Roche Diagnostics GmbH (Roche Holding AG), University of Utah Research Foundation (State of Utah)
• Inventors: Gutekunst, Martin, Wittwer, Carl T., Lohmann, Sabine
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/281,448
• Time in Patent Office: 931 Days
• Field of Search: 435/6, 435/21.2, 435/39, 536/24.3, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio

Abstract

The invention provides a new method for the quantification of a nucleic acid, wherein in a first step, the nucleic acid is amplified by an amplifying agent. Subsequently, first, second or n th order derivatives is calculated. The obtained value can then be used to calculate the initial concentration of the analyte.

Claims

1. A method for quantification of the concentration of a nucleic acid in a nucleic acid sample, comprising the steps of:

a) contacting said nucleic acid sample with an amplifying agent;

b) amplifying at least one predetermined locus of the nucleic acid in said nucleic acid sample to create a nucleic acid amplification product;

c) determining the amount of the nucleic acid amplification product as a function of amplification reaction time;

d) calculating the first, second or n th order derivative of said function, wherein n is a natural number;

e) determining the maximum of said derivative; and

f) calculating from said maximum the initial concentration of the nucleic acid in said nucleic acid sample.

2. The method of claim 1, wherein during one phase of the amplification reaction the amount of amplification product increases progressively and wherein after said progressive phase, the rate of amplification decreases.

3. The method of claim 1, wherein the amount of the amplification product is determined during an exponential phase of the amplification.

4. The method of claim 1, wherein the amplification product is detected by means of fluorescence.

5. The method of claim 2, wherein the amplification is obtained by a polymerase chain reaction and the amplification product is detected by a double-stranded DNA binding entity.

6. The method of claim 4, wherein the amplification product is detected by at least one fluorescently labeled polynucleotide probe the sequence of which is identical with or complementary to the sequence of the amplification product over a range of at least 10 contiguous residues.

7. The method of claim 4 wherein amplification is obtained by a polymerase chain reaction and the amplification product is detected by two polynucleotide probes, each labeled with a fluorescent entity, such that when both probes are hybridized to one strand of the nucleic acid amplification product, fluorescence resonance energy transfer occurs between the two fluorescent entities.

9. The method of claim 3 wherein said derivative is calculated by a mathematical fit.

10. The method of claim 9 wherein the amplification product is detected by means of fluorescence.

11. The method of claim 9 wherein the amplification is obtained by a polymerase chain reaction and the amplification product is detected by a double-stranded binding entity.

12. The method of claim 1 wherein the step of calculating the first, second or n th order derivative of said function comprises calculating the second derivative.

13. The method of claim 1 wherein the step of calculating the first, second or n th order derivative of said function comprises calcuilating, the first derivative.

8. A method for quantification of the concentration of a nucleic acid in a nucleic acid sample, comprising the steps of:

a) contacting said nucleic acid sample with an amplifying agent;

b) amplifying at least one predetermined locus of the nucleic acid in said nucleic acid sample to create a nucleic acid amplification product;

c) determining the amount of the amplification product as a function of amplification reaction time;

d) assembling a first data set representing the amount of the nucleic acid amplification product at different reaction times;

e) performing a polynomial fit on subsets of said first data set;

f) assambling from said popynomial fit a second data set representing smoothened kinetics of generation of the amplification product at different reaction times;

g) obtaining a nathematical function from subsets of said second data set;

h) calculating the first, second or n th order derivative of said function, wherein n is a natural number;

i) determining the maximum of said derivative; and

j) calculating from said maximum the initial concentration of the nuicleic acid in said nucleic acid sample.

14. A method for quantification of the concentration of a nucleic acid in a nucleic acid sample, comprising the steps of:

a) contacting said nucleic acid sample with an amplifyingy agent;

b) amplifying at least one predetermined locus of the nucleic acid in said nucleic acid sample to create a nucleic acid amplification product;

c) determining the amount of the nucleic acid amplification product as a function of amplification reaction time;

d) calculating the second derivative of said function;

e) determining the maximum of said second derivative; and

f) calculating from said maximun the initial concentration of the nucleic acid in said nucleic acid sample.

15. A method for quantification of the concentration of a nucleic acid in a nucleic acid sample, comprising the steps of:

a) contacting said nucleic acid sample with an amplifying agent;

b) amplifying at least one predetermined locus of the nucleic acid in said nucleic acid sample by a process comprising the step cf subjecting the sample to a number of amplification cycles to create a nucleic acid amplification product;

c) determining a value corresponding to the relative amount of the nucleic acid amplification product for each amplification cycle to generate a data set;

d) generatinig a function from said data set;

e) calculating an n th order derivative of said function, wherein n is an integer;

f) determining a fractional cycle number corresponding to a maximum of said derivative;

g) obtaining a standard curve generated using steps a-e on a plurality of additional nucleic acid samples, each additional sample having a known concentration of the nucleic acid; and

h) determining the initial concentration of the nucleic acid in said nucleic acid sample using the standard curve.