Claims for Patent: 6,063,568

Summary for Patent: 6,063,568

Title:	Quantitation of RNA transcripts using genomic DNA as the internal amplification competitor
Abstract:	A method for the quantitative monitoring of gene expression without either co-amplification of an added template or use of an endogenous constitutive transcript is provided. The process 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/intron sequences for the mRNA/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 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. The invention may be used, for example, in the determination of gene expression in both research and commercial applications.
Inventor(s):	Gerdes; John C. (Denver, CO), Marmaro; Jeffrey M. (Aurora, CO)
Assignee:	Molecular Innovations, Inc. (Denver, CO)
Application Number:	08/850,613
Patent 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 primer and/or probe sequences specific for mRNA transcripts and endogenous genomic DNA; c) detecting mRNA transcripts and endogenous genomic DNA by: (I) hybridizing said probe sequences to said mRNA transcripts and endogenous genomic DNA; or (ii) amplifying both said mRNA transcripts and endogenous genomic DNA using said primer sequences to produce amplimers; d) quantitatively determining 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. 2. The method of claim 1 wherein said nucleic acid is a transcribed gene in which both the mRNA and endogenous genomic DNA are characterized sequences and possess sequence heterogeneity. 3. The method of claim 1 wherein said amplification of endogenous genomic DNA and mRNA transcripts is a competitive reaction, said endogenous genomic DNA being a quantitation standard. 4. The method of claim 1 wherein in step c(i) a first detection probe that is complementary to and hybridizes to DNA intron sequences and a second probe that is complementary to and hybridizes to mRNA sequences at the splice junction are used to measure DNA intron sequences relative to mRNA sequences at the splice junction. 5. The method of claim 1 wherein said endogenous genomic DNA and mRNA transcripts are amplified using a single set of primers and labeled during amplification with a plurality of probes labeled with detectable moieties. 6. The method of claim 5 wherein said primers are complementary to or hybridize to sequences within two exons, one of said probes is hybridized to said mRNA sequence at the splice junction and one of said plurality of probes is hybridized to an intron sequence of said DNA. 7. The method of claim 5 wherein one or both of said primers is chemically modified or labeled. 8. The method of claim 1 wherein said detecting step comprises duplex amplification using a common primer capable of amplifying both genomic DNA and expressed mRNA from the same gene sequence. 9. The method of claim 8 wherein said probes are labeled with detectable moieties and the common primer is either chemically modified or labeled or is not chemically modified or labeled. 10. The method of claim 8 further comprising a DNA primer complementary to an intron sequence and a mRNA primer complementary to a DNA sequence at the intron splice junction sequence, said primers simultaneously detecting gene expression. 11. The method of claim 1 wherein said determination comprises the separation of amplimers by size. 12. The method of claim 11 wherein said separation comprises chromatography. 13. The method of claim 12 wherein said chromatography comprises gel electrophoresis, size-exclusion chromatography or high-performance liquid chromatography (HPLC). 14. The method of claim 1 wherein said probes are labeled with reporter molecules or haptens. 15. The method of claim 1 wherein said determination comprises sequence heterogeneity discrimination. 16. A method for quantitation of gene expression which comprises: a) extracting nucleic acid from a biological specimen; b) designing primer and probe sequences for amplifying mRNA and endogenous genomic DNA by means of hybridization to the same primer recognition sequences; c) performing RT-PCR with said primer and probe sequences using a single tube, one step reaction wherein distinguishable fluorescent signals specific to mRNA or genomic DNA are generated; and d) determining relative gene expression through multiplex componenting the ratio of fluorescent signals or determining absolute mRNA copy number relative to a standard curve based on known concentrations of purified nucleic acid sequence of a gene. 17. A method for quantitation of gene expression which comprises: a) extracting nucleic acid from a biological specimen; b) designing primer and probe sequences for amplifying mRNA and endogenous genomic DNA by means of the same primer recognition sequences; c) amplifying both mRNA and endogenous genomic DNA via primer-based amplification methodology yielding mRNA and DNA amplimers; d) specifically hybridizing and quantifying endogenous genomic DNA and mRNA amplimers using labeled capture probes and e) quantitatively determining gene expression as expressed by the ratio of mRNA amplimers to endogenous genomic DNA amplimers. 18. The method of claim 17 further comprising detecting capture probes using a label on one or a plurality of said primers.

Details for Patent 6,063,568

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2016-05-02
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2016-05-02
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2016-05-02