Quantitation of nucleic acids using the polymerase chain reaction
AssigneeHoffmann-La Roche Inc.
InventorsAlice M. Wang, Michael V. Doyle, David F. Mark
Life of this patent
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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 or 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.
Claims18
A plasmid for use as an internal standard for quantitation of at least one target nucleic acid sequence contained within a sample which plasmid comprises:
- a standard nucleic acid segment comprising a 5' sequence and a 3' sequence which sequences provide upstream and downstream primer hybridization sites in said plasmid which primer hybridization sites are identical to upstream and downstream primer hybridization sites within said target nucleic acid sequence such that a primer pair, comprising an upstream oligonucleotide primer and a downstream oligonucleotide primer, will function in a PCR reaction to amplify said standard nucleic acid segment and said target nucleic acid segment, wherein upon amplification said standard and said target segments can be distinguished by size or by use of an internal oligonucleotide probe.
A kit for the quantitation of a target nucleic acid segment in a biological sample comprising individual containers which provide:
- a predetermined initial amount of an internal standard nucleic acid segment for quantitation of a target nucleic acid wherein said internal standard binds the same primers as are bound by said target nucleic acid segment;
- and
- an oligonucleotide primer pair wherein said primer pair can serve to amplify said internal standard and said target nucleic acid.
An amplification reaction mixture for the quantitation of a target nucleic acid segment in a biological sample, said reaction mixture comprising:
- said target nucleic acid;
- a predetermined initial amount of an internal standard nucleic acid segment for quantitation of a target nucleic acid, wherein said internal standard binds the same primers as are bound by said target nucleic acid segment;
- and
- an oligonucleotide primer pair wherein said primer pair can serve to amplify said internal standard and said target nucleic acid, wherein following amplification said standard and target amplified nucleic acid segments are distinguishable by size or by use of internal hybridization probes.
A reverse transcription reaction mixture for reverse transcribing a target mRNA suspected of being present in a biological sample, said reaction mixture comprising a predetermined initial amount of internal standard cRNA, a target RNA, and a target-specific primer for initiating cDNA synthesis, wherein said primer can serve to initiate reverse transcription of a nucleic acid segment contained within said standard cRNA together with a segment contained within the particular target nucleic acid, and wherein said standard segment is further distinguished by having a downstream hybridization site identical in sequence to a downstream by hybridization site in said target nucleic acid, whereby following reverse transcription the resulting target and standard cDNAs can serve as templates for amplification for providing standard and target amplified nucleic acid segments which are distinguishable by size or by use of internal hybridization probes.
Dependencies are inferred from the claim text.