Nucleic acid amplification method: ramification-extension amplification method (RAM)

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

• (a) contacting the nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequence in the nucleic acid with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable amplification probe 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 amplification probe, wherein the circularizable amplification probe is bound on its 3′ and 5′ ends to the adjacent but not the overlapping sequences in the target nucleic acid;
• (b) separating the complex from unbound circularizable amplification probes and unbound target nucleic acids and washing the complex;
• (c) ligating the 3′ and 5′ ends of the circularizable amplification probe with a ligating agent that joins nucleotide sequences such that a circular amplification probe is formed;
• (d) amplifying the circular amplification probe of step (c) by contacting the circular amplification probe of step (c) with an extension primer that is complementary and hybridizable to generic portions of the circular amplification probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby said extension primer is extended around the circular amplification probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular amplification probe;
• and
• (e) detecting the single stranded DNA, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

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

• (a) contacting the nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequences in the nucleic acid with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable amplification probe having 3′ and 5′ regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, said 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 amplification probe, wherein the circularizable amplification probe is bound on its 3′ and 5′ ends to the adjacent but not the overlapping sequences in the target nucleic acid;
• (b) ligating the 3′ and 5′ ends of the circularizable amplification probe with a ligating agent that joins nucleotide sequences such that a circular amplification probe is formed;
• (c) amplifying the circular amplification probe of step (b) by contacting the circular amplification probe of step (b) with a first extension primer that is complementary and hybridizable to generic portions of the circular amplification probe and a second extension primer that is substantially identical to the generic portions of the circular amplification probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the first extension primer is extended around the circular amplification probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular amplification 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
• (d) detecting the extension products, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

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

• (a) contacting the nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequences in the nucleic acid with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable amplification probe 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 amplification probe, wherein the circularizable amplification probe is bound on its 3′ and 5′ ends to the adjacent but not the overlapping sequences in the target nucleic acid;
• (b) ligating the 3′ and 5′ ends of the circularizable amplification probe with a ligating agent that joins nucleotide sequences such that a circular amplification probe is formed;
• (c) amplifying the circular amplification probe of step (b) by contacting the circular amplification probe of step (b) with an extension primer that is complementary and hybridizable to generic portions of the circular amplification probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby said extension primer is extended around the circular amplification probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular amplification probe;
• and
• (d) detecting the single stranded DNA, wherein detection thereof indicates the presence of the target nucleic acid in the sample.