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US 8,628,923 B2Granted
Single cell nucleic acid analysis
AssigneeFluidigm Corporation
InventorsAmy Hamilton, Min Lin, Alain Mir, Martin Pieprzyk
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Abstract
The present invention provides methods for analysis of genomic DNA and/or RNA from small samples or even single cells. Methods for analyzing genomic DNA can entail whole genome amplification (WGA), followed by preamplification and amplification of selected target nucleic acids. Methods for analyzing RNA can entail reverse transcription of the desired RNA, followed by preamplification and amplification of selected target nucleic acids.
Claims35
A method for analyzing DNA from a single cell, said method comprising:
- (a) performing whole genome amplification of the genome of a single cell to produce an amplified genome, wherein the method entails performing whole genome amplification for fewer than, or about, 10 polymerase chain reaction (PCR) cycles;
- (b) preamplifying the amplified genome using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids;
- (c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification;
- and
- (d) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers.
2 more elements
A method for analyzing mRNA from a single cell, said method comprising:
- (a) preparing DNA from the mRNA of a single cell;
- (b) preamplifying the DNA using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids, wherein said preamplification is carried out for 18 polymerase chain reaction (PCR) cycles or fewer;
- (c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification;
- and
- (d) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers, and wherein the amplicons are detected by a method selected from a real-time quantitation method, monitoring the amount of amplification product after a predetermined number of cycles, and melting curve analysis.
2 more elements
A method for analyzing non-coding RNA from a single cell, said method comprising:
- (a) preparing DNA from the non-coding RNA of a single cell;
- (b) preamplifying the DNA using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids;
- (c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification;
- and
- (d) amplifying and detecting the plurality of amplicons wherein different amplicons are amplified and detected in separate chambers, and wherein the amplicons are detected by a method selected from a real-time quantitation method, monitoring the amount of amplification product after a predetermined number of cycles, and melting curve analysis.
2 more elements
A method for analyzing DNA from a single cell, said method comprising:
- (a) performing whole genome amplification of the genome of a single cell to produce an amplified genome, wherein the whole genome amplification comprises polymerase chain reaction (PCR) cycles;
- (b) preamplifying the amplified genome using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids;
- (c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification;
- and
- (d) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers, and wherein primer pairs used in preamplification and/or amplification can amplify and detect single nucleotide polymorphisms (SNPs), by a method selected from a real-time quantitation method, monitoring the amount of amplification product after a predetermined number of cycles, and melting curve analysis.
2 more elements
A method for analyzing RNA from a single cell, said method comprising:
- (a) preparing DNA from the RNA of a single cell;
- (b) preamplifying the DNA using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids;
- (c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification;
- and
- (d) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers, and wherein primer pairs used in preamplification and/or amplification can amplify and detect single nucleotide polymorphisms (SNPs), by a method selected from a real-time quantitation method, monitoring the amount of amplification product after a predetermined number of cycles, and melting curve analysis.
2 more elements
Dependencies are inferred from the claim text.