Circularizable nucleic acid probes and amplification methods

An amplification kit comprising:

• (a) at least one first spanning oligonucleotide;
• (b) at least one circularizable probe;
• (c) at least one extension primer;
• and
• (d) at least one second spanning oligonucleotide.

A method for producing linear circularizable probes comprising:

• (a) contacting a first spanning oligonucleotide under conditions that allow nucleic acid hybridization between complementary sequences in the spanning oligonucleotide with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable probe having 3′ and 5′ regions that are complementary to adjacent but not overlapping sequences in the spanning oligonucleotide, such that a complex is formed comprising the spanning oligonucleotide and the circularizable probe, wherein the circularizable probe is bound on its 3′ and 5′ ends to the adjacent but not overlapping sequences in the spanning oligonucleotide;
• (b) ligating the 3′ and 5′ ends of the circularizable probe with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (c) amplifying the circular probe of step (b) by contacting the circular probe of step (b) with an extension primer that is complementary and hybridizable to the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe;
• (d) cleaving the single stranded DNA of repeating units with a restriction enzyme, under conditions whereby the restriction enzyme cleaves the single stranded DNA of repeating units into single unit length oligonucleotides;
• (e) contacting a second spanning oligonucleotide, comprising nucleic acid sequences complementary to the unit length oligonucleotide, under conditions that allow nucleic acid hybridization between complementary sequences in the second spanning oligonucleotide with at least one unit length oligonucleotide of step (d);
• and
• (f) repeating steps (b) through (d) such that linear circularizable probes are formed.

A method of detecting a target nucleic acid in a sample comprising:

• (a) contacting a first spanning oligonucleotide under conditions that allow nucleic acid hybridization between complementary sequences in the spanning oligonucleotide with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable probe having 3′ and 5′ regions that are complementary to adjacent but not overlapping sequences in the spanning oligonucleotide, such that a complex is formed comprising the spanning oligonucleotide and the circularizable probe, wherein the circularizable probe is bound on its 3′ and 5′ ends to the adjacent but not overlapping sequences in the spanning oligonucleotide;
• (b) ligating the 3′ and 5′ ends of the circularizable probe with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (c) amplifying the circular probe of step (b) by contacting the circular probe of step (b) with an extension primer that is complementary and hybridizable the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe;
• (d) cleaving the single stranded DNA of repeating units with a restriction enzyme, under conditions whereby the restriction enzyme cleaves the single stranded DNA of repeating units into single unit length oligonucleotides;
• (e) contacting a second spanning oligonucleotide, comprising nucleic acid sequences complementary to the unit length oligonucleotide, under conditions that allow nucleic acid hybridization between complementary sequences in the second spanning oligonucleotide with at least one unit length oligonucleotide of step (d);
• (f) repeating steps (b) through (d);
• (g) contacting the first spanning oligonucleotide under conditions that allow nucleic acid hybridization between complementary sequences in the first spanning oligonucleotide with at least one unit length oligonucleotide produced from step (f);
• (h) ligating the 3′ and 5′ ends of the unit length oligonucleotide produced from step (g) with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (i) contacting the target nucleic acid under conditions that allow nucleic acid hybridization between complementary sequences in the target nucleic acid with at least one circular probe produced from step (h), the circular probe comprising regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, the complementary regions separated by a generic region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, such that a complex is formed comprising the target nucleic acid and the circular probe;
• (j) amplifying the circular probe of the complex of step (i) by contacting the circular probe of the complex of step (i) with an extension primer that is complementary and hybridizable to the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe;
• and
• (k) detecting the single stranded DNA of repeating units, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

A method of detecting a target nucleic acid in a sample comprising:

• (a) contacting a first spanning oligonucleotide under conditions that allow nucleic acid hybridization between complementary sequences in the spanning oligonucleotide with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable probe having 3′ and 5′ regions that are complementary to adjacent but not overlapping sequences in the spanning oligonucleotide, such that a complex is formed comprising the spanning oligonucleotide and the circularizable probe, wherein the circularizable probe is bound on its 3′ and 5′ ends to the adjacent but not overlapping sequences in the spanning oligonucleotide;
• (b) ligating the 3′ and 5′ ends of the circularizable probe with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (c) amplifying the circular probe of step (b) by contacting the circular probe of step (b) with an extension primer that is complementary and hybridizable to the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe;
• (d) cleaving the single stranded DNA of repeating units with a restriction enzyme, under conditions whereby the restriction enzyme cleaves the single stranded DNA of repeating units into single unit length oligonucleotides;
• (e) contacting a second spanning oligonucleotide, comprising nucleic acid sequences complementary to the unit length oligonucleotide, under conditions that allow nucleic acid hybridization between complementary sequences in the second spanning oligonucleotide with at least one unit length oligonucleotide of step (d);
• (f) repeating steps (b) through (d);
• (g) contacting the first spanning oligonucleotide under conditions that allow nucleic acid hybridization between complementary sequences in the first spanning oligonucleotide with at least one unit length oligonucleotide produced from step (f);
• (h) ligating the 3′ and 5′ ends of the unit length oligonucleotide produced from step (g) with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (i) contacting the target nucleic acid under conditions that allow nucleic acid hybridization between complementary sequences in the target nucleic acid with at least one circular probe produced from step (h), the circular probe comprising regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, the complementary regions separated by a generic region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, such that a complex is formed comprising the target nucleic acid and the circular probe;
• (j) amplifying the circular probe of the complex of step (i) by contacting the circular probe of the complex of step (i) with a first extension primer that is complementary and hybridizable to the circular probe, a second extension primer that is substantially identical to portions of the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe, and multiple copies of the second extension primer hybridize to complementary regions of the single stranded DNA and are extended by the DNA polymerase to provide extension products;
• and
• (k) detecting the extension products, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

A method of detecting a target nucleic acid in a sample comprising:

• (a) contacting a first spanning oligonucleotide under conditions that allow nucleic acid hybridization between complementary sequences in the spanning oligonucleotide with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable probe having 3′ and 5′ regions that are complementary to adjacent but not overlapping sequences in the spanning oligonucleotide, such that a complex is formed comprising the spanning oligonucleotide and the circularizable probe, wherein the circularizable probe is bound on its 3′ and 5′ ends to the adjacent but not overlapping sequences in the spanning oligonucleotide;
• (b) ligating the 3′ and 5′ ends of the circularizable probe with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (c) amplifying the circular probe of step (b) by contacting the circular probe of step (b) with an extension primer that is complementary and hybridizable to the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe;
• (d) cleaving the single stranded DNA of repeating units with a restriction enzyme, under conditions whereby the restriction enzyme cleaves the single stranded DNA of repeating units into single unit length oligonucleotides;
• (e) contacting a second spanning oligonucleotide, comprising nucleic acid sequences complementary to the unit length oligonucleotide, under conditions that allow nucleic acid hybridization between complementary sequences in the second spanning oligonucleotide with at least one unit length oligonucleotide of step (d);
• (f) repeating steps (b) through (d) such that linear circularizable probes are formed;
• (g) contacting the target nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequences in the target nucleic acid with at least one circularizable probe produced in step (f) having 3′ and 5′ regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, the 3′ and 5′ regions separated by a generic region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, such that a complex is formed comprising the target nucleic acid and the circularizable probe, wherein the circularizable probe is bound on its 3′ and 5′ ends to the adjacent but not overlapping sequences in the target nucleic acid;
• (h) ligating the 3′ and 5′ ends of the circularizable probe utilized in step (g) with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (i) amplifying the circular probe of step (h) by contacting the circular probe of step (h) with an extension primer that is complementary and hybridizable to the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe;
• and
• (j) detecting the single stranded DNA of repeating units, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

A method of detecting a target nucleic acid in a sample comprising:

• (a) contacting a first spanning oligonucleotide under conditions that allow nucleic acid hybridization between complementary sequences in the spanning oligonucleotide with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable probe having 3′ and 5′ regions that are complementary to adjacent but not overlapping sequences in the spanning oligonucleotide, such that a complex is formed comprising the spanning oligonucleotide and the circularizable probe, wherein the circularizable probe is bound on its 3′ and 5′ ends to the adjacent but not the overlapping sequences in the spanning oligonucleotide;
• (b) ligating the 3′ and 5′ ends of the circularizable probe with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (c) amplifying the circular probe of step (b) by contacting the circular probe of step (b) with an extension primer that is complementary and hybridizable to the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe;
• (d) cleaving the single stranded DNA of repeating units with a restriction enzyme, under conditions whereby the restriction enzyme cleaves the single stranded DNA of repeating units into single unit length oligonucleotides;
• (e) contacting a second spanning oligonucleotide, comprising nucleic acid sequences complementary to the unit length oligonucleotide, under conditions that allow nucleic acid hybridization between complementary sequences in the second spanning oligonucleotide with at least one unit length oligonucleotide of step (d);
• (f) repeating steps (b) through (d) such that linear circularizable probes are formed;
• (g) contacting the target nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequences in the target nucleic acid with at least one circularizable probe produced in step (f) having 3′ and 5′ regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, the 3′ and 5′ regions separated by a generic region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, such that a complex is formed comprising the target nucleic acid and the circularizable probe, wherein the circularizable probe is bound on its 3′ and 5′ ends to the adjacent but not overlapping sequences in the target nucleic acid;
• (h) ligating the 3′ and 5′ ends of the circularizable probe utilized in step (g) with a ligating agent that joins nucleotide sequences such that a circular probe is formed;
• (i) amplifying the circular probe of step (h) by contacting the circular probe of step (h) with a first extension primer that is complementary and hybridizable to the circular probe, a second extension primer that is substantially identical to portions of the circular probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the extension primer is extended around the circular probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular probe, and multiple copies of the second extension primer hybridize to complementary regions of the single stranded DNA and are extended by the DNA polymerase to provide extension products;
• and
• (j) detecting the extension products, wherein detection thereof indicates the presence of the target nucleic acid in the sample.