Quantitation of RNA transcripts using genomic DNA as the internal amplification competitor

• US 6,258,543 B1
• Filed: 09/17/1999
• Issued: 07/10/2001
• Est. Priority Date: 05/02/1996
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Xtrana Inc. (Bio-Techne Corporation)
• Inventors: Marmaro, Jeffrey M., Gerdes, John C.
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US09/398,539
• Time in Patent Office: 662 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 435/91.51, 536/24.33
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2539/105 Involving introns, exons, or splice junctions
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2545/107 with a competitive internal standard/control
• CPC Class Codes: C12Q 2561/101 Taqman

Abstract

This invention is directed to methods for the quantitative measurement of specific gene expression levels in biological samples. In one embodiment, methods for the quantitative monitoring of gene expression without either co-amplification of an added template or use of an endogenous constitutive transcript are provided. The former involves a duplex amplification reaction in which a single set of primers is used to amplify both genomic DNA and expressed mRNA from the same gene sequence. These primers are targeted for sequences flanking the splice junction and intron sequences for the mRNA and DNA respectively. By their use, any suitable nucleic acid amplification technology yields mRNA and DNA amplimers which are distinguishable by length and sequence heterogeneity. These amplimers are also present in the final amplification reaction in ratios which are dependent upon the ratios of the expressed mRNA to the DNA in the sample, allowing the quantitation of mRNA in a sample which is normalized to the number of copies of genomic DNA since the genomic DNA acts as the internal quantitation standard, and in effect yields the amount of mRNA per cell. Any detection methodology which can detect amplimers of different lengths or sequences can be used for post amplification quantitation. This strategy may be employed for any gene system in which the mRNA sequence differs from the original genomic DNA sequence. In another embodiment, methods for the quantitative monitoring of gene expression involving determining the ratio of genomic material to expressed mRNA without nucleic acid amplification and/or primer binding are described. This method includes the independent and direct detection of the genomic material and mRNA by complementary probe binding without prior amplification. Additional methods for quantitative measurement of gene expression rates without RNA transcription region introns are described. The invention is useful in gene expression determination in research and commercial applications.

Claims

1. A method for quantitation of gene expression which comprises:

a) extracting nucleic acid from a biological specimen wherein said nucleic acid comprises mRNA transcripts and endogenous genomic DNA;

b) designing complementary probe sequences specific for said mRNA transcripts and said endogenous genomic DNA, wherein said probe sequences discriminate between said endogenous geinomic DNA and said mRNA transcripts by sequence heterogeneity;

c) hybridizing said complementary probe sequences to said mRNA transcripts and endogenous genomic DNA;

d) directly detecting said endogenous genomic DNA and said mRNA transcripts; and

e) quantitatively determining gene expression as expressed by the ratio of said mRNA transcripts to said endogenous genomic DNA.

2. The method as defined in claim 1, wherein direct detection of said endogenous genomic DNA and mRNA transcripts is accomplished via signal amplification methodologies or direct probe detection.

3. A method for quantitation of gene expression which comprises:

a) extracting nucleic acid from a biological specimen, wherein said nucleic acid comprises mRNA transcripts and endogenous genomic DNA;

b) designing sets of primer sequences specific for said mRNA transcripts and endogenous genomic DNA, wherein said sets of primer sequences include at least two primers which specifically hybridize to a coding region of said mRNA transcript and said endogenous genomic DNA, and wherein said sets of primers further include a primer which hybridizes to sequences of said endogenous genomic DNA but does not hybridize to sequences of said mRNA transcript;

c) amplifying both said mRNA transcripts and endogenous genomic DNA using said primer sequences to produce mRNA amplimers and endogenous genomic DNA amplimers in a competitive reaction, said endogenous genomic DNA being a quantitation standard;

d) detecting said mRNA transcript amplimers and said endogenous genomic DNA amplimers; and

e) quantitatively dctermining gene expression as expressed by the ratio of mRNA transcripts to endogenous genomic DNA or as expressed by the ratio of mRNA transcript amplimers to endogenous genomic DNA amplimers.

4. The method as defined in claim 3, wherein said primers are labeled with detectable moieties.

5. The method as defined in claim 3 wherein said determination comprises the separation of said mRNA transcript amplimers and said endogenous genomic DNA amplimers by size.

6. The method as defined in claim 5 wherein said separation comprises chromatography.

7. The method as defined in claim 6, wherein said chromatography comprises gel electrophoresis, size-exclusion chromatography or high-performance liquid chromatography (HPLC).

8. The method as defined in claim 3, wherein said quantitation is determined by a change in gene transcription rate, wherein said change in gene transcription rate is expressed as the ratio of (mRNA amplimers plus endogenous genomic DNA amplimers) to endogenous genomic DNA amplimers.

9. The method as defined in claim 3, wherein the nucleic acid is the Estrogen Receptor gene having a transcription start site.

10. The method as defined in claim 9, wherein the primer sets comprise a common reverse primer that is complementary to a first sequence located in Exon 1 in the mRNA transcript, a first forward primer that is complementary to a second sequence located in Exon 1 in the mRNA transcript, and a second forward primer that is complementary to a sequence located in an intron upstream of the transcription start site.

11. The method of claim 3, wherein said primers include a first forward primer that is complementary to a sequence in said endogenous genomic DNA, a second forward primer that is complementary to a sequence in said mRNA transcript, and a common reverse primer, said reverse primer capable of amplifying both said mRNA transcript and said endogenous genomic DNA and acting as a competitive amplification primer.

12. The method of claim 3, wherein said detection comprises:

a) designing complementary probe sequences specific for said mRNA amplimers and said endogenous genomic DNA amplimers;

b) hybridizing said complementary probe sequences to said amplimers and said endogenous genomic DNA amplimers; and

c) detecting said hybridized probes.

13. The method of claim 12, wherein said detection comprises fluorescence detection, electro-chemiluminescence detection, chemiluminescence detection, bioluminescence detection and enzyme-lined immunosorbent assay (ELISA) detection.