Homogeneous amplification and detection of nucleic acids

A method for amplifying and detecting a target polynucleotide sequence of a polynucleotide analyte, which comprises:

• (a) providing in combination a sample suspected of containing a polynucleotide analyte having said target polynucleotide sequence, reagents for amplifying said polynucleotide analyte to produce copies of said target polynucleotide sequence, a first oligonucleotide probe having nucleotide sequences S1 and S2 and a second oligonucleotide probe having sequences S3 and S4 wherein each of S1, S2, S3 and S4 is about 8 to 25 nucleotides in length and wherein the sequences comprising at least one of the probes are linked such that either (i) they are non-contiguous and can bind to contiguous or non-contiguous sites on one of the strands of said copies or (ii) the sites to which they hybridize on one of the strands of said copies are noncontiguous and wherein said probes (i) do not hybridize to said copies during said amplifying and (ii) subsequent to said amplifying, both of said probes can hybridize to one of the strands of said copies, and (iv) each of said probes is comprised of a label that facilitates detection of said probes hybridized to said strands,
• (b) subjecting said combination to conditions for amplifying said polynucleotide analyte,
• (c) after step (b) subjecting said combination to conditions under which both of said probes hybridize to one of said strands to form a termolecular complex, and
• (d) detecting said complex.

A method of detecting a target polynucleotide containing a target polynucleotide sequence wherein all reagents required for said method are first combined with said target polynucleotide, said method comprising:

• (a) dissociating said target polynucleotide sequence into single strands when said target polynucleotide sequence is double stranded,
• (b) hybridizing an oligonucleotide primer to the 3'-end of each of said single strands,
• (c) extending said primer hybridized to each of said single strands along the single strand to produce a copy of said target polynucleotide sequence,
• (d) dissociating said copy into single strands,
• (e) hybridizing two oligonucleotide probes to one of said single strands wherein at least one of said probes is comprised of two sequences that hybridize with one of said single strands, wherein said sequences either (i) are non-contiguous and can bind to contiguous or non-contiguous sites on said strand or (ii) the sites on said strand to which said sequences hybridize are non-contiguous,
• (f) detecting the binding of both of said probes to said single strand, the presence thereof being related to the presence of said target polynucleotide.

A method for detecting a target sequence of a target polynucleotide ("target sequence"), said method comprising:

• (a) amplifying said target sequence by a method comprising:
• (i) hybridizing to the 3'-end of said target sequence a first oligonucleotide primer ("first primer"),
• (ii) extending, in the presence of a polymerase and nucleotide triphosphates, said first primer along at least said target sequence to produce an extended first primer, said first primer hybridizing to, and being extended along, (1) said extended first primer or (2) an extended second oligonucleotide primer ("second primer") wherein said extended second primer results from the extension of a second primer that hybridizes to and is extended along a polynucleotide that is complementary (complementary polynucleotide) to said target sequence,
• (iii) dissociating said extended first primer from said target sequence,
• (iv) hybridizing, to the 3'-end of said extended first primer, said first or said second primer,
• (v) extending said first or said second primer along said extended first primer,
• (vi) dissociating said extended first primer or said extended second primer from said extended first primer,
• (vii) hybridizing, to the 3'-end of said extended first or said extended second primer, said first primer, and
• (viii) repeating steps (v)-(vii), and
• (b) detecting said extended first primer and/or said extended second primer by means of a first oligonucleotide probe having nucleotide sequences S1 and S2 and a second oligonucleotide probe having sequences S3 and S4, wherein S1, S2, S3 and S4 are each about 10 to 20 nucleotides in length wherein the sequences comprising at least one of the probes are linked such that its two sequences either (i) are non-contiguous and can bind to contiguous or non-contiguous sites on one of said extended primers or (ii) hybridize to sites on one of said extended primers that are noncontiguous and wherein said probes (A) are present during said amplifying, (B) do not hybridize to said extended first and/or second primers during said amplifying of step (a) and (C) do not interfere with said amplifying of step (a) and (D) subsequent to said amplifying of step (a), both of said probes can hybridize to one of said extended first and/or said extended second primers to form a termolecular complex, and (E) one or both of said probes contains a label that facilitates detection of said probes hybridized to said extended first and/or said extended second primers.

A kit for use in an amplification and detection of a target polynucleotide sequence of a target polynucleotide, said kit comprising in packaged combination:

• (a) an oligonucleotide primer which hybridizes to said target polynucleotide and is extended along said target polynucleotide sequence to produce extended oligonucleotide primer,
• (b) nucleoside triphosphates,
• (c) a nucleotide polymerase,
• (d) a first oligonucleotide probe having nucleotide sequences S1 and S2, and
• (e) a second oligonucleotide probe having sequences S3 and S4,
• wherein S1, S2, S3 and S4 are each about 10 to 20 nucleotides in length and
• wherein the sequences comprising at least one of said first or said second oligonucleotide probes are linked such that either (i) they are non-contiguous and can bind to contiguous or non-contiguous sites on said extended polynucleotide primer or a complementary sequence thereto or (ii) the sites to which they hybridize on said extended polynucleotide primer or a complementary sequence thereto are noncontiguous, and
• wherein said probes have the characteristics that they (i) do not hybridize to said extended oligonucleotide primer during said amplification and (ii) subsequent to said amplification, both of said first and second oligonucleotide probes can hybridize to said extended oligonucleotide primer or said complementary sequence, and (iii) one or both of said probes contain a label that facilitates detection of said probes hybridized to said extended oligonucleotide primer or said complementary sequence.

A method for amplifying and detecting a target polynucleotide sequence, which comprises:

• (a) providing in combination a sample suspected of containing a target polynucleotide having said target polynucleotide sequence, reagents for amplifying said target polynucleotide sequence to produce copies thereof, a first oligonucleotide probe and a second oligonucleotide probe wherein said probes (i) do not hybridize to said copies during said amplifying and (ii) do not interfere with said amplifying and (iii) subsequent to said amplifying, both of said probes can hybridize to one the strands of said copies, and wherein at least one said probes is associated with a particle,
• (b) subjecting said combination to conditions for amplifying said target polynucleotide sequence to produce said copies,
• (c) thereafter subjecting said combination to conditions under which both of said probes hybridize to one of said strands and result in agglutination of said particles, and
• (d) detecting said agglutination wherein the presence of agglutination indicates the presence of said target polynucleotide sequence.