Quantitation of RNA transcripts using genomic DNA as the internal amplification competitor
First Claim
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.
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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.
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Citations
13 Claims
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1. A method for quantitation of gene expression which comprises:
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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. - View Dependent Claims (2)
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3. A method for quantitation of gene expression which comprises:
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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. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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.
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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.
Specification