Quantitation of nucleic acids using the polymerase chain reaction

• US 5,219,727 A
• Filed: 09/28/1989
• Issued: 06/15/1993
• Est. Priority Date: 08/21/1989
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Doyle, Michael V., Wang, Alice M., Mark, David F.
• Primary Examiner(s): Moskowitz, Margaret
• Assistant Examiner(s): Fleisher, Mindy B.
• Application Number:
  US07/413,623
• Time in Patent Office: 1,356 Days
• Field of Search: 435/6, 435/810, 435/91, 536/27, 935/77, 935/78, 436/501, 436/808
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2545/107 with a competitive internal standard/control
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The present invention provides a method for determining the amount of a target acid segment in a sample by polymerase chain reaction. The method involves the simultaneous amplification of the target nucleic acid segment and an internal standard nucleic acid segment. The amount of amplified DNA from each segment is determined and compared to standard curves to determine the amount of the target nucleic acid segment present in the sample prior to amplification. The method is especially preferred for determining the quantity of a specific mRNA species in a biological sample. Additionally, an internal standard is provided useful for quantitation of multiple mRNA species.

Claims

1. A method for quantifying a target nucleic acid segment in a sample, which method comprises the steps of:

(a) adding to said sample a predetermined initial amount of standard nucleic acid segment wherein said standard nucleic acid segment binds to same primers as are bound by said target nucleic acid segment in a reaction mixture;

(b) treating said sample under conditions suitable for carrying out a polymerase chain reaction, wherein said nucleic acids are rendered single-stranded and exposed to an agent for polymerization, deoxynucleoside 5'"'"' triphosphates, and a pair of oligonucleotide primers, wherein said pair of primers is specific for both the target and standard nucleic acid segments, such that an extension product of each primer of said pair can be synthesized using separate strands of the target and standard segments as a template for synthesis, such that the extension product of one primer, when it is separated from the template strand, can serve as a template for the synthesis of the extension product of the other primer of said pair wherein said amplified target and standard segments are distinguishable by size or by the use of internal probes, wherein said internal probes may be differentially labeled for each of said amplified target and standard segments;

(c) separating the primer extension product from the templates on which they were synthesized to form single-stranded molecules;

(d) repeating steps (b) and (c) on the single stranded molecules produced in step (c) at least once, whereby each repeat of steps (b) and (c) is one amplification cycle;

(e) measuring the amounts of the amplified target and standard segments produced in step (d); and

(f) calculating from the amplified target and standard segments produced in step (d) the amount of said target nucleic acid segment present in the sample before amplification.

2. The method of claim 1 further comprising the step of preparing at least one dilution from the mixture of step (a) prior to step (b), whereby the amount of standard and target nucleic acid segments prior to said amplification reaction are varied.

3. The method of claim 1 wherein from 10 to 50 amplification cycles are performed, and wherein samples of said reaction mixture are removed and used to determine through which cycles the reaction is in exponential phase.

4. The method of claim 1 wherein said target nucleic acid segment is an mRNA molecule;

said standard nucleic acid is a cRNA molecule; and

said RNAs are reverse transcribed into cDNA molecules following step (a) and prior to step (b).

5. The method of claim 4 wherein the nucleic acid sequence of said cRNA molecule is synthesized using a plasmid template selected from the group AW108 and AW109.

6. The method of claim 4 wherein the pair of oligonucleotide primers of step (b) is labeled, and the amounts of amplified target and standard segments produced are measured according to step (e) by determining the amount of label incorporated into each of said amplified nucleic acid segments.

7. The method of claim 6 wherein said target nucleic acid segment is contained within an mRNA sequence, wherein said mRNA sequence encodes a lymphokine.

8. The method of claim 6 wherein said target nucleic acid segment is contained within a nucleic acid sequence, wherein said nucleic acid sequence encodes a protein selected from the group consisting of TNF, M-CSF, PDGF-A, PDGF-B, PDGF-R, apo-E, LDL-R, HMG, IL-1, IL-2, LPL, G-CSF, GM-CSF, aFGF, bFGF, c-fms, TGF-β

, LFA-1, IL-2Rα

, α

-actin, desmin, β

-actin, IL-6, IFN-α

, IFN-γ

, IL-6R, PDGF-α

R, IL-2Rβ

, IL-3, IL-4, and HIV proteins.

9. The method of claim 8 wherein said sample is a human blood cell sample or a human arterial vessel sample.

10. The method of claim 8 wherein said pair of oligonucleotide primers are selected from the group consisting of:

space="preserve" listing-type="tabular">______________________________________ 5'"'"'-CAGAGGGAAGAGTTCCCCAG-3'"'"', 5'"'"'-CCTTGGTCTGGTAGGAGACG-3'"'"';

5'"'"'-GAACAGTTGAAAGATCCAGTG-3'"'"', 5'"'"'-TCGGACGCAGGCCTTGTCATG-3'"'"';

5'"'"'-CCTGCCCATTCGGAGGAAGAG-3'"'"', 5'"'"'-TTGGCCACCTTGACGCTGCG-3'"'"';

5'"'"'-GAAGGAGCCTGGGTTCCCTG-3'"'"', 5'"'"'-TTTCTCACCTGGACAGGTCG-3'"'"';

5'"'"'-TTCCTGGCAGGATGCCAGGC-3'"'"', 5'"'"'-GGTCAGTTGTTCCTCCAGTTC-3'"'"';

5'"'"'-CAATGTCTCACCAAGCTCTG-3'"'"', 5'"'"'-TCTGTCTCGAGGGGTAGCTG-3'"'"';

5'"'"'-TACCATGTCAGGGGTACGTC-3'"'"', 5'"'"'-CAAGCCTAGAGACATAATCATC-3'"'"';

5'"'"'-GTCTCTGAATCAGAAATCCTTCTATC-3'"'"', 5'"'"'-CATGTCAAATTTCACTGCTTCATCC-3'"'"';

5'"'"'-AAACAGATGAAGTGCTCCTTCCAGG-3'"'"', 5'"'"'-TGGAGAACACCACTTGTTGCTCCA-3'"'"';

5'"'"'-GAATGGAATTAATAATTACAAGAATCCC-3'"'"', 5'"'"'-TGTTTCAGATCCCTTTAGTTCCAG-3'"'"';

5'"'"'-TGACCACCCAGCCATCCTTC-3'"'"', 5'"'"'-GAGGAGGTGTTGACTTCATTC-3'"'"'; and

5'"'"'-GAGATTTCTCTGTATGGCACC-3'"'"', 5'"'"'-CTGCAAATGAGACACTTTCTC-3'"'"'. ______________________________________