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.
Claims37
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 amplification 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 a plurality of target nucleic acids;
- (c) distributing the plurality of amplicons within a device;
- and
- (d) amplifying and detecting the plurality of amplicons in the device.
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 a plurality of target nucleic acids, wherein said preamplification is carried out for 18 amplification cycles or fewer;
- (c) distributing the plurality of amplicons within a device;
- and
- (d) amplifying and detecting the plurality of amplicons in the device.
The method of claims 1 or 34, wherein said whole genome amplification is performed for more than two amplification cycles.
The method of claim 34, wherein said whole genome amplification is performed for fewer than, or about, 10 amplification cycles.
The method of claims 1 or 34, wherein said whole genome amplification is carried out using a technique selected from the group consisting of primer extension PCR (PEP), degenerated oligonucleotide primed PCR, ligation-mediated PCR (LMP), the T7-based linear amplification of DNA (TLAD), and multiple displacement amplification (MDA).
The method of claim 35, wherein the RNA comprises non-coding RNA.
The method of claims 14 or 33, wherein the non-coding RNA is selected from the group consisting of small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), and Piwi-interacting RNAs (piRNA).
The method of claims 14 or 33, wherein DNA is produced from the non-coding RNA by reverse transcription or amplification.
(canceled)
The method of claims 1 or 35, wherein said preamplification is carried out for 8-18 cycles.
The method of claims 1, 2 or 33-35, wherein said plurality of target-specific primer pairs used for preamplification amplifies one or more single nucleotide polymorphisms (SNPs).
(canceled)
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 plurality of amplicons within a device;
- and
- (d) amplifying and detecting the plurality of amplicons in the device.
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 plurality of amplicons within a device;
- and
- (d) amplifying and detecting the plurality of amplicons in the device.
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 plurality of amplicons within a device;
- and
- (d) amplifying and detecting the plurality of amplicons in the device.
Dependencies are inferred from the claim text.