Methods, systems, and compositions for counting nucleic acid molecules
AssigneeProgenity, Inc.
InventorsMatthew Sekedat, Jeffrey Buis, Ronald David Beaubien, Sharat Singh, Jeff Perry
Life of this patent
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Abstract
Compositions and methods, systems, and kits for detecting and quantifying variations in numbers of molecules, particularly variations in gene dosage, e.g., due to gene duplication, or to variations from the normal euploid complement of chromosomes, e.g., trisomy of one or more chromosomes that are normally found in diploid pairs, without digital sequencing.
Claims127
A method for counting target molecules on a solid support, comprising:
- a) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support;
- b) detecting formation of the at least one complex in a process comprising:
- i) extending the primer in the complex in a rolling circle amplification (RCA) reaction to form RCA product;
- ii) hybridizing a plurality of labeled probes to the RCA product;
- and
- iii) detecting hybridized labeled probe;
- wherein hybridized labeled probe is indicative of the presence of the target molecule on the solid support.
A method for counting target molecules on a solid support, comprising:
- a) providing a silanized surface comprising at least one of
- acrylic groups;
- reactive amine groups;
- b) forming a plurality of complexes on the glass surface, the plurality of complexes comprising at least one of:
- an RCA product comprising a plurality of hybridized labeled probes;
- a double-stranded scaffold product comprising a plurality of concatemerized labeled scaffold oligonucleotides;
- wherein formation of a complex is indicative of the presence of a target molecule on the glass surface, and wherein forming said plurality of complexes comprises exposing the glass surface to a solution comprising graphene oxide;
- and
- c) counting the plurality of complexes.
A composition comprising a silanized surface bound to a plurality of complexes, each comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to a solid support, and a reaction mixture comprising
- at least 0.2 units per μL, preferably at least 0.8 units per μL of Phi29 DNA polymerase;
- a buffer
- at least 400 μM, preferably at least 600 μM, more preferably at least 800 μM total dNTPs;
- PEG, preferably at least 2 to 10% (w:v), preferably at least 12%, preferably at least 14%, preferably at least 16%, preferably at least 18% to 20% PEG.
A composition comprising a silanized surface bound to a plurality of complexes each comprising an RCA product comprising a plurality of hybridized labeled probes, and a solution comprising graphene oxide.
The method of claim 123, wherein the diagnostic purpose comprises detecting an aneupoloidy.
Dependencies are inferred from the claim text.