Methods for quantitating DNA using digital multiple displacement amplification
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology
InventorsPaul Blainey, Liyi Xu
Life of this patent
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Abstract
The present invention relates to methods of quantifying, amplifying, or preparing nucleic acid molecules, where the methods involve contacting a sample to be tested with nucleic acid molecule amplification reaction components and a label to form a reaction sample. The methods further involve partitioning the reaction sample into droplets or a gel and allowing nucleic acid molecule amplification to occur.
Claims24
A method of quantifying a nucleic acid molecule, the method comprising:
- (a) contacting a sample to be tested with degenerate oligonucleotide primers, a DNA polymerase, and a label that detects nucleic acid molecules to form a reaction sample;
- (b) partitioning the reaction sample at limiting dilution into monodisperse droplets, wherein each droplet corresponds to a single nucleic acid molecule amplification reaction comprising a nucleic acid molecule or no nucleic acid molecule;
- (c) allowing the nucleic acid molecule amplification reaction to occur in the droplets;
- and
- (d) determining the number of droplets comprising nucleic acid molecules by detecting the label;
- thereby quantifying the nucleic acid molecule in the sample to be tested.
A method of amplifying a nucleic acid molecule in a sample, the method comprising:
- (a) contacting a sample with degenerate oligonucleotide primers, a polymerase, and a label that detects nucleic acid molecules to form a reaction sample;
- (b) partitioning the reaction sample at limiting dilution into a hydrogel;
- and
- (c) allowing the amplification reaction to occur in the hydrogel.
A method of preparing nucleic acid molecules for analysis, the method comprising:
- (a) contacting a sample to be tested with degenerate oligonucleotide primers, a DNA polymerase, and a label that detects nucleic acid molecules to form a reaction sample;
- (b) partitioning the reaction sample into monodisperse droplets, wherein each droplet corresponds to a single nucleic acid amplification reaction comprising a nucleic acid molecule or no nucleic acid molecule;
- and
- (c) allowing the nucleic acid amplification reaction to occur in the droplets.
Dependencies are inferred from the claim text.