Methods, systems, and compositions for counting nucleic acid molecules
OwnerMatthew Sekedat, Jeffrey Buis +3inventors
InventorsMatthew Sekedat, Jeffrey Buis, Ronald David Beaubien, Jr., Sharat Singh, Jeff Perry
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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.
Claims21
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, wherein forming the RCA product comprises extending the primer bound to the surface on the solid support on a circularized nucleic acid probe in a reaction mixture comprising:
- at least 0.2 units per μL of Phi29 DNA polymerase;
- a buffer;
- at least 400 μM total dNTPs;
- and
- at least 12% PEG, wherein the PEG has an average molecular weight between 400 and 800;
- 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 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 of Phi29 DNA polymerase;
- a buffer;
- at least 400 μM total dNTPs;
- at least 12% PEG, wherein the PEG has an average molecular weight between 400 and 800.
Dependencies are inferred from the claim text.