Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-05-02
2000-05-16
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 435 9121, 435 9151, 536 2433, C12Q 168, C12P 1934, C07H 2104
Patent
active
060635681
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.
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Gerdes John C.
Marmaro Jeffrey M.
Bernard Julie L.
Molecular Innovations, Inc.
Myers Carla J.
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