A method of solid phase nucleic acid hybridization assay incorporating a luminescent label

Claim 2

An improved method of nucleic acid hybridization assay comprising: allowing solid carrier bound three-component hybrid molecules to form by complementary base pairing of the following: (1) single-stranded sample nucleic acid to be assayed contained in a liquid sample of measurable volume; (2) single-stranded first fragment of nucleic acid having a nucleotide sequence of at least 15 bases and which is attached to a solid carrier, said first fragment of nucleic acid being capable of hybridizing with said sample nucleic acid; and (3) single-stranded second fragment of nucleic acid having a nucleotide sequence of at least 15 bases and to which is attached a luminescent label or a luminescent label acceptor to which a luminescent label may be attached subsequent to allowing the three-component hybrid molecules to form, said second fragment of nucleic acid being capable of hybridizing with said sample nucleic acid but not capable of hybridizing with said first fragment of nucleic acid; and separating (1) the solid carrier bound three-component hybrid molecules to which luminescent label or luminescent label acceptor is attached from (2) luminescent label or luminescent label acceptor not attached to said solid carrier bound three-component hybrid molecules; wherein the improvement comprises: allowing complementary base pairing between the sample nucleic acid and the first fragment of nucleic acid to occur while the solid carrier comprising a plurality of water insoluble particles of about 50 microns or less in diameter, to which the first fragment of nucleic acid is attached, is in a substantially suspended state; and measuring the luminescence of luminescent label attached to the three-component hybrid molecules while the solid carrier, to which such molecules are attached, has been concentrated by microfiltration to a volume substantially less than the volume of the liquid sample.

Claim 3

The method of claim 1 wherein the three-component hybrid molecules are allowed to form by first, allowing hybridization of the first fragment of nucleic acid with the sample nucleic acid while the solid carrier, to which the first fragment of nucleic acid is attached, is in a substantially suspended state, forming two-component hybrid molecules; and second, allowing three-component hybrid molecules to form by complementary base pairing of the second fragment of nucleic acid with the sample nucleic acid contained in the two-component hybrid molecules.

Claim 4

3 . The method of claim 1 wherein the three-component hybrid molecules are allowed to form by simultaneous complementary base pairing of the sample nucleic acid, the first fragment of nucleic acid, and the second fragment of nucleic acid, while the solid carrier, to which the first fragment of nucleic acid is attached, is in a substantially suspended state.

Claim 5

An improved method of nucleic acid hybridization assay comprising allowing two species of solid carrier bound two-component hybrid molecules to form by complementary base pairing of the following: (1) single-stranded sample nucleic acid to be assayed contained in a liquid sample of measurable volume; (2) single-stranded first fragment of nucleic acid having a nucleotide sequence of at least 15 bases and which is attached to a solid carrier, said first fragment of nucleic acid being capable of hybridizing with said sample nucleic acid; and (3) single-stranded second fragment of nucleic acid having a nucleotide sequence of at least 15 bases and to which is attached a luminescent label or a luminescent label acceptor to which a luminescent label may be attached subsequent to allowing the two species of solid carrier bound two-component hybrid molecules to form, said second fragment of nucleic acid being capable of hybridizing with said first fragment of nucleic acid but not capable of hybridizing with said sample nucleic acid; and separating (1) the two species of solid carrier bound two-component hybrid molecules to which luminescent label or luminescent label acceptor is attached from (2) luminescent label or luminescent label acceptor not attached to said two species of solid carrier bound two-component hybrid molecules; wherein the improvement comprises: allowing complementary base pairing between (1) the first fragment of nucleic acid and (2) the sample nucleic acid or the second fragment of nucleic acid to occur while the solid carrier comprising a plurality of water insoluble particles of about 50 microns or less in diameter, to which the first fragment of nucleic acid is attached, is in a substantially suspended state; and measuring the luminescence of the luminescent label attached to two-component hybrid molecules while the solid carrier, to which such molecules are attached, has been concentrated by microfiltration to a volume substantially less than the volume of the liquid sample.

Claim 6

An improved method of nucleic acid hybridization assay comprising allowing two species of solid carrier bound two-component hybrid molecules to form by complementary base pairing of the following: (1) single-stranded sample nucleic acid to be assayed contained in a liquid sample of measurable volume; (2) single-stranded first fragment of nucleic acid having a nucleotide sequence of at least 15 bases and which is attached to a solid carrier, said first fragment of nucleic acid being capable of hybridizing with said sample nucleic acid; and (3) single-stranded second fragment of nucleic acid having a nucleotide sequence of at least 15 bases and to which is attached a luminescent label or a luminescent label acceptor to which a luminescent label may be attached subsequent to allowing the two species of solid carrier bound two-component hybrid molecules to form, said second fragment of nucleic acid being capable of hybridizing with said first fragment of nucleic acid but not capable of hybridizing with said sample nucleic acid; and separating (1) the two species of solid carrier bound two-component hybrid molecules to which luminescent label or luminescent label acceptor is attached from (2) luminescent label or luminescent label acceptor not attached to said two species of solid carrier bound two-component hybrid molecules; wherein the improvement comprises: allowing complementary base pairing between (1) the first fragment of nucleic acid and (2) the sample nucleic acid or the second fragment of nucleic acid to occur while the solid carrier comprising a plurality of water insoluble particles of about 50 microns or less in diameter, to which the first fragment of nucleic acid is attached, is in a substantially suspended state; and measuring the luminescence of the luminescent label attached to two-component hybrid molecules while the solid carrier, to which such molecules are attached, has been concentrated by microfiltration to a volume substantially less than the volume of the liquid sample.

Claim 7

The method of claim 4 wherein the two species of solid carrier bound two-component hybrid molecules are allowed to form by first, allowing hybridization of the sample nucleic acid with an excess of first fragment of nucleic acid while the solid carrier, to which the first fragment of nucleic acid is attached, is in a substantially suspended state; and second, allowing hybridization of (1) first fragment of nucleic acid which was not hybridized by the sample nucleic acid, with (2) the second fragment of nucleic acid.

Claim 8

The method of claim 4 wherein the two species of solid carrier bound two-component hybrid molecules are allowed to form by first, allowing hybridization of the first fragment of nucleic acid with the second fragment of nucleic acid while the solid carrier, to which the first fragment of nucleic acid is attached, is in a substantially suspended state; and second, allowing displacement of such hybridized second fragment of nucleic acid by hybridization of the first fragment of nucleic acid with the sample nucleic acid.

Claim 9

The method of claim 4 wherein the two species of solid carrier bound two-component hybrid molecules are allowed to form by simultaneous complementary base pairing of (1) sample nucleic acid and (2) second fragment of nucleic acid in competition for first fragment of nucleic acid, while the solid carrier, to which the first fragment of nucleic acid is attached, is in a substantially suspended state.

Claim 10

The method of claim 4 wherein the two species of solid carrier bound two-component hybrid molecules are allowed to form by first, allowing hybridization of sample nucleic acid with the first fragment of nucleic acid, while the solid carrier, to which the first fragment of nucleic acid is attached, is substantially suspended; and second, allowing competition for such first fragment of nucleic acid by hybridization of second fragment of nucleic acid with such first fragment of nucleic acid.

Claim 11

An improved method of nucleic acid hybridization assay comprising allowing two species of two-component hybrid molecules, one species bound to solid carrier and the other species not bound, to form by complementary base pairing of the following: (1) single-stranded sample nucleic acid to be assayed contained in a liquid sample of measurable volume: (2) single-stranded first fragment of nucleic acid having a nucleotide sequence of at least 15 bases and which is attached to a solid carrier, said first fragment of nucleic acid not being capable of hybridizing with said sample nucleic acid; and (3) single-stranded second fragment of nucleic acid having a nucleotide sequence of at least 15 bases and to which is attached a luminescent label or a luminescent label acceptor to which a luminescent label may be attached subsequent to allowing the two species of two-component hybrid molecules to form, said second fragment of nucleic acid being capable of hybridizing with said sample nucleic acid or said first fragment of nucleic acid; and separating the species of solid carrier bound two-component hybrid molecules from the species of unbound two-component hybrid molecules; wherein the improvement comprises: allowing complementary base pairing between (1) the first fragment of nucleic acid and (2) the second fragment of nucleic acid to occur while the solid carrier comprising a plurality of water insoluble particles of about 50 microns or less in diameter, to which the first fragment of nucleic acid is attached, is in a substantially suspended state; and measuring the luminescence of the luminescent label attached to solid carrier bound two-component hybrid molecules while the solid carrier, to which such molecules are attached, has been concentrated by microfiltration to a volume substantially less than the volume of the liquid sample.

Claim 12

An improved method of nucleic acid hybridization assay comprising allowing two species of two-component hybrid molecules, one species bound to solid carrier and the other species not bound, to form by complementary base pairing of the following: (1) single-stranded sample nucleic acid to be assayed contained in a liquid sample of measurable volume: (2) single-stranded first fragment of nucleic acid having a nucleotide sequence of at least 15 bases and which is attached to a solid carrier, said first fragment of nucleic acid not being capable of hybridizing with said sample nucleic acid; and (3) single-stranded second fragment of nucleic acid having a nucleotide sequence of at least 15 bases and to which is attached a luminescent label or a luminescent label acceptor to which a luminescent label may be attached subsequent to allowing the two species of two-component hybrid molecules to form, said second fragment of nucleic acid being capable of hybridizing with said sample nucleic acid or said first fragment of nucleic acid; and separating the species of solid carrier bound two-component hybrid molecules from the species of unbound two-component hybrid molecules; wherein the improvement comprises: allowing complementary base pairing between (1) the first fragment of nucleic acid and (2) the second fragment of nucleic acid to occur while the solid carrier comprising a plurality of water insoluble particles of about 50 microns or less in diameter, to which the first fragment of nucleic acid is attached, is in a substantially suspended state; and measuring the luminescence of the luminescent label attached to solid carrier bound two-component hybrid molecules while the solid carrier, to which such molecules are attached, has been concentrated by microfiltration to a volume substantially less than the volume of the liquid sample.

Claim 13

The method of claim 9 wherein the two species of two-component hybrid molecules are allowed to form by simultaneous complementary base pairing of (1) sample nucleic acid and (2) first fragment of nucleic acid in competition for second fragment of nucleic acid, while the solid carrier, to which the first fragment of nucleic acid is attached, is in a substantially suspended state.