Sampling method and apparatus for amplification reaction analysis
AssigneeSention, Inc.
InventorVladimir I. Slepnev
Life of this patent
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Abstract
The invention provides sampling methods that permit the quantitative analysis of nucleic acid amplification reactions. Quantitative information garnered during an amplification regimen permits the development of a detailed amplification profile that in turn permits the reliable determination of original template abundance. The methods disclosed are particularly useful for quantitative expression analysis of multiple genes or transcription units, both in the same amplification reaction and in multiple amplification reactions.
Claims63
A method of analyzing a nucleic acid amplification:
- providing a nucleic acid amplification reaction mixture comprising a plurality of different amplification templates;
- subjecting said reaction mixture to an amplification regimen;
- dispensing or withdrawing an aliquot from said reaction mixture at plural stages during said amplification regimen;
- separating and detecting nucleic acids in said aliquot;
- determining the quantity of a plurality of separated nucleic acid species in said aliquot;
- and
- for each said separated nucleic acid species from each said stage, correlating the quantity of said species with the stage at which said aliquot comprising said species was dispensed, wherein said correlating generates an amplification profile of said nucleic acid amplification.
A method of analyzing the expression of a plurality of RNA transcripts between first and second gene expressing entities, said method comprising
- providing a first nucleic acid amplification reaction mixture, said mixture comprising a plurality of different amplification templates, wherein said amplification templates comprise reverse transcription products from a plurality of RNA transcripts from a first gene expressing entity;
- providing a second nucleic acid amplification reaction mixture, said mixture comprising a plurality of different amplification templates, wherein said amplification templates comprise reverse transcription products from a plurality of RNA transcripts from a second gene expressing entity;
- subjecting said reaction mixtures to an amplification regimen;
- dispensing or withdrawing an aliquot from said first and said second reaction mixtures at plural stages during said amplification regimen;
- separating and detecting nucleic acids in said aliquot;
- determining the quantity of a plurality of separated nucleic acid species in said aliquot;
- for each said separated nucleic acid species from each said stage, correlating the quantity of said species with the stage at which said aliquot comprising said species was dispensed, thereby generating a transcriptional profile of said plurality of RNA transcripts expressed by said first and said second gene expressing entities;
- and
- comparing said transcriptional profile from said first gene expressing entity with said transcriptional profile from said second gene expressing entity.
A method of monitoring the amplification of a nucleic acid sequence, the method comprising:
- providing a nucleic acid amplification reaction mixture comprising a template of said nucleic acid sequence;
- performing an amplification regimen on the mixture;
- automatically dispensing an aliquot of said reaction mixture at plural stage intervals throughout the amplification regimen;
- separating and detecting the nucleic acid species in said aliquot;
- and,
- for respective ones of plural separated species, determining the quantity of separated nucleic acid in said aliquot.
A method of determining the transcription profile of a nucleic acid sequence, the method comprising:
- providing a nucleic acid amplification reaction mixture;
- performing an amplification regimen on the mixture;
- dispensing an aliquot of said reaction mixture at plural stage intervals throughout the amplification regimen;
- separating and detecting the nucleic acid species in said aliquot;
- and,
- for respective ones of plural separated species, determining the quantity of separated nucleic acid in said aliquot;
- and
- determining the transcription profile of said nucleic acid sequence.
Dependencies are inferred from the claim text.