Patent ID: 6569647
Filing Date: 2003-05-27
Classification: C12Q,Y10T

Abstract:
A method for in situ detection of a target nucleic acid in a sample, comprising the steps of:(a) preparing a tissue sample from a histological specimen to be analyzed for the presence of a target nucleic acid; (b) washing said tissue sample; (c) adding a circularizable amplification probe having 3â€² and 5â€² regions that are complementary to adjacent but noncontiguous sequences in the target nucleic acid, said 3â€² and 5â€² regions separated by a linker region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, wherein said circularizable amplification probe contains at least one ligand, such that a complex is formed comprising the target nucleic acid and the circularizable probe in the presence of paramagnetic particles coated with a ligand binding moiety that recognizes and binds to said ligand on said amplification probe; (d) ligating the 3â€², and 5â€² ends of said circularizable probe with a ligating agent that joins nucleotide sequences such that a circular amplification probe is formed; (e) washing the complex; (f) amplifying said circular amplification probe by contacting said complex with a first extension primer that is complementary and hybridizable to a portion of the linker region of the circular amplification probe and a second extension primer that is substantially identical to a portion of the linker region of the circular amplification probe that does not overlap with the portion of the linker region to which the first extension probe is complementary, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the first extension primer is extended around the circle 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 whereby the extension products of the second extension primers displace downstream copies of the second extension primers and corresponding extension products to provide displaced single strands to which multiple copies of said first extension primer bind and are extended by the DNA polymerase; (g) allowing said amplification to proceed until multiple copies of double stranded amplified DNA of varying lengths are produced; and (h) detecting said amplified DNA, wherein detection thereof indicates the presence of the target nucleic acid in the clinical sample.