Restriction/ligation labeling for primer initiated multiple copying of DNA ssequences

Claim 1

A method for amplifying dsDNA comprising a target sequence., said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a 5'-3' first strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced or substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer binding site sequence proximal to the 3'-terminus of said labeled single stranded DNA and complementary sequence on the 5'-terminus; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.

Claim 2

A method for amplifying dsDNA comprising a target sequence., said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a 5'-3' first strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced or substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer binding site sequence proximal to the 3'-terminus of said labeled single stranded DNA and complementary sequence on the 5'-terminus; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.

Claim 9

A method for amplifying dsDNA comprising a target sequence, said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a first 5'-3' strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced or substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer binding site sequence proximal to the 3'-terminus of said labeled single stranded DNA and complementary sequence on the 5'-terminus; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence by hybridizing said single stranded labeled DNA with a probe having a nucleic acid sequence complementary to said target sequence and comprising a member of a specific binding pair to produce probe bound strands, combining said probe bound strands with the complementary member of said specific binding pair bound to a solid support, and separating single stranded labeled DNA bound to said support from unbound single stranded labeled DNA; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.

Claim 10

A method for amplifying dsDNA comprising a target sequence, said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a first 5'-3' strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced or substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer binding site sequence proximal to the 3'-terminus of said labeled single stranded DNA and complementary sequence on the 5'-terminus; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence by hybridizing said single stranded labeled DNA with a probe having a nucleic acid sequence complementary to said target sequence and comprising a member of a specific binding pair to produce probe bound strands, combining said probe bound strands with the complementary member of said specific binding pair bound to a solid support, and separating single stranded labeled DNA bound to said support from unbound single stranded labeled DNA; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.

Claim 13

A method for amplifying dsDNA comprising a target sequence, said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a first 5'-3' strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced and substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer sequence proximal to the 3'-terminus of said labeled single stranded DNA and its complementary sequence 5' to said single stranded DNA; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence by hybridizing said single stranded labeled DNA with a probe having a nucleic acid sequence complementary to said target sequence and comprising a solid support to produce probe bound strands, and separating single stranded labeled DNA bound to said support from unbound single stranded labeled DNA; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.

Claim 14

A method for amplifying dsDNA comprising a target sequence, said method comprising: combining a dsDNA sample with labeling DNA moieties in registry with termini of restriction fragments of said sample dsDNA, wherein said labeling DNA moieties comprise at least one primer binding site sequence, said labeling DNA moieties comprise a first 5'-3' strand and a ligaid or truncated ligaid strand, at least partially complementary to said first strand, and said restriction fragments are formed prior to, during, or after said combining; covalently joining both strands of the labeling DNA to the associated strands of said dsDNA in register, with the proviso that said truncated ligaid may be displaced and substituted with a ligaid strand comprising said primer binding site sequence prior to said covalent joining; denaturing said dsDNA labeled strands to produce labeled single stranded DNA comprising a primer sequence proximal to the 3'-terminus of said labeled single stranded DNA and its complementary sequence 5' to said single stranded DNA; substantially separating any single stranded labeled DNA comprising said target sequence from single stranded labeled DNA lacking said target sequence by hybridizing said single stranded labeled DNA with a probe having a nucleic acid sequence complementary to said target sequence and comprising a solid support to produce probe bound strands, and separating single stranded labeled DNA bound to said support from unbound single stranded labeled DNA; and amplifying said single stranded labeled DNA comprising said target sequence employing primers complementary to said primer binding site sequence.