Solid phase nucleic acid target capture and replication using strand displacing polymerases

A method for the purification and replication of target nucleic acids (1) from a sample, comprising:

• a) contacting said sample with reverse strand displacement primer oligonucleotides (3) and incubating to anneal the reverse strand displacement primer oligonucleotides (3) to target-specific sequences (18) on the target nucleic acids (1) whereby said incubating is performed in a buffer solution and not on a solid support (4);
• b) contacting said sample with reverse primer/capture probe oligonucleotides (2) having a 5′ tail comprising a sequence that can be used for amplification and incubating to anneal the reverse primer/capture probe oligonucleotides (2) to target-specific sequences (17) on the target nucleic acids (1) that are located on the 5′ side of the sequences (18) to which the reverse strand displacement primer oligonucleotides (3) hybridize to form complexes of reverse primer/capture probe oligonucleotides (2) and reverse strand displacement primer oligonucleotides (3) bound to the target nucleic acids (1);
• c) purifying the formed complexes on a solid support (4) by capturing the complexes and washing away excess reverse primer/capture probe oligonucleotides (2) and reverse strand displacement primer oligonucleotides (3) that are not bound to the target nucleic acids (1) and sample components other than the complexes;
• d) after the step of purifying said formed complexes, extending the reverse primer/capture probe oligonucleotides (2) with 5′ tail on the target nucleic acids (1) to form strands (5), and extending the reverse strand displacement primer oligonucleotides (3) on the target nucleic acids (1) with a nucleic acid strand displacing polymerase, wherein extension of the reverse strand displacement primer oligonucleotides (3) displaces, from the target nucleic acids (1), the extended reverse primer/capture probe oligonucleotides (2) to give single-stranded copies (5), which are copies of the target nucleic acids (1) and which are attached to the solid support (4);
• e) washing the double-stranded copies (6) resulting from the extension of reverse strand displacement primer oligonucleotides (3) that are not attached to the solid support (4) from the solid support (4);
• f) contacting a medium containing the single stranded copies (5) bound to the solid support (4) with solution containing a mixture of forward primers (7) with a 5′ tail comprising a sequence that can be used for amplification, that hybridize to target-specific sequences (20) on the single stranded copies (5), and forward strand displacement primers (8) that hybridize to target-specific sequences (19) on the single stranded copies (5), wherein the sequence (19) to which the forward strand displacement primers (8) hybridize is located on the 3′ side of the sequences (20) to which the forward primers (7) with a 5′ tail hybridize on the single stranded copies (5), and incubating to anneal forward primers (7) and forward strand displacement primers (8) to single stranded copies (5) to form complexes and washing away excess forward primers (7) and forward strand displacement primers (8) that are not bound to the single stranded copies (5);
• g) after washing away the excess forward primers (7) and forward strand displacement primers (8) extending forward primers (7) with tail 5′ and the forward strand displacement primers (8) hybridized on the single stranded copies (5) immobilized on the solid support (4) with a nucleic acid strand displacing polymerase, wherein extension of the strand displacement primers (8) displaces the single-stranded extension products (9) of the forward primers (7) with 5′ tail from the single stranded copies (5) and from the solid support (4);
• h) recovering the single-stranded extension products (9) in the supernatant solution bathing the solid support (4) said single-stranded extension products (9) containing the tail 5′ tail sequence of the forward primers (7) and the complement of the 5′ tail of the reverse primer/capture probe oligonucleotides (2), and adding them to with an amplification reagent mix;
• and
• i) amplifying the extended forward primer strands (9) using one or both of the attached tail 5′ sequences.

A method for the purification and replication of target nucleic acids (1) from a sample, comprising:

• a) contacting said sample with reverse primer/capture probe oligonucleotides (2) having a 5′ tail comprising a sequence that can be used for amplification and incubating to anneal the reverse primer/capture probe oligonucleotides (2) to target-specific sequences (17) on the target nucleic acids (1);
• b) purifying said target nucleic acids (1) on a solid support (4) by capturing the complexes and washing away and sample components other than the complexes;
• c) hybridizing reverse strand displacement primer oligonucleotides (3) to target-specific sequences (18) on the target nucleic acids (1) that are located on the 3′ side of the sequences (17) to which the reverse primer/capture probe oligonucleotides (2) hybridize;
• d) washing the solid support (4) to remove excess strand displacement oligonucleotides (3) that are not bound to the target nucleic acids (1);
• e) extending the reverse primer/capture probe oligonucleotides (2) with 5′ tail on the target nucleic acids (1) to form strands (5), and extending the reverse strand displacement primer oligonucleotides (3) on the target nucleic acids (1) with a nucleic acid strand displacing polymerase, wherein extension of the reverse strand displacement primer oligonucleotides (3) displaces, from the target nucleic acids (1), the extended reverse primer/capture probe oligonucleotides (2) to give single-stranded copies (5), which are copies of the target nucleic acids (1) and which are attached to the solid support (4);
• f) washing the double-stranded copies (6) resulting from the extension of reverse strand displacement primer oligonucleotides (3) from the solid support (4);
• g) contacting a medium containing the single stranded copies (5) bound to the solid support (4) with solution containing a mixture of forward primers (7) with a 5′ tail comprising a sequence that can be used for amplification, that hybridize to target-specific sequences (20) on the single stranded copies (5), and forward strand displacement primers (8) that hybridize to target-specific sequences (19) on the single stranded copies (5), wherein the sequence (19) to which the forward strand displacement primers (8) hybridize is located on the 3′ side of the sequence (20) to which the forward primers (7) with a 5′ tail hybridize on the single stranded copies (5), and incubating to anneal forward primers (7) and forward strand displacement primers (8) to single stranded copies (5) to form complexes and washing away excess forward primers (7) and forward strand displacement primers (8) that are not bound to the single stranded copies (5);
• h) after washing away the excess forward primers (7) and forward strand displacement primers (8) extending forward primers (7) with tail 5′ and the forward strand displacement primers (8) hybridized on the single stranded copies (5) immobilized on the solid support (4) with a nucleic acid strand displacing polymerase, wherein extension of the strand displacement primers (8) displaces the single-stranded extension products (9) of the forward primers with 5′ tail (7) from the single stranded copies (5) and from the solid support (4);
• i) recovering the single-stranded extension products (9) in the supernatant solution bathing the solid support (4) said single-stranded extension products (9) containing the tail 5′ tail sequence of the forward primers (7) and the complement of the 5′ tail of the reverse primer/capture probe oligonucleotides (2), and adding them to with an amplification reagent mix;
• and
• j) amplifying the extended forward primer strands (9) using one or both of the attached tail 5′ sequences.