Methods, systems, and compositions for counting nucleic acid molecules
AssigneeProgenity, Inc.
InventorsMatthew Sekedat, Jeffrey Buis, Ronald David Beaubien, JR., 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.
Claims36
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 surface on 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.
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A composition comprising a silanized surface on a solid support bound to a plurality of complexes, each comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe, wherein the primer is bound to the solid support, wherein:
- i) the primer is covalently linked to the silanized surface;
- or
- ii) the primer comprises a biotin moiety and the silanized surface comprises avidin or streptavidin,
- 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.
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A composition comprising a silanized glass surface bound to a plurality of complexes each comprising an RCA product comprising a plurality of hybridized labeled probes comprising fluorescent labels, and a solution comprising graphene oxide, wherein the plurality of complexes is bound in an irregular dispersal.
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Dependencies are inferred from the claim text.