US 2006/0188896 A1 · Nucleic acid amplification with integrated multiplex dectection

Abstract

A method mediated with in-vitro transcription (“IVT”) which permits miniaturization of multiplexed DNA and RNA analysis, and in which elongation-mediated multiplexed analysis of polymorphisms (eMAP®) is used as the analysis step, is described. Also described is a method mediated with IVT is for selecting a designated strand from T7-tagged double stranded DNA: wherein, the selected strand forms the template for RNA synthesis. In one embodiment, double stranded DNA incorporating the T7 (or other) promoter sequence at the 3′ end or the 5′ end is produced, for example, by amplification of genomic DNA using the Polymerase Chain Reaction (PCR). Also disclosed are nested PCR designs permitting allele analysis in combination with strand selection by IVT. Further, in one embodiment of a homogeneous format for transcription-mediated amplification and multiplexed detection (which may be particularly suited for viral or pathogen detection), encoded microparticles display “looped” capture probe configurations permitting the generation of a signal upon capture of RNA product and real-time assay monitoring.

Claims47

Dependent claims

A method of performing nucleic acid analysis using in-vitro transcription amplification of the target nucleic acid molecules to obtain amplification of the target nucleic acid molecules of about 103, comprising:

amplifying the target nucleic acid molecules with in vitro transcription (wherein in the amplification reaction mRNA is reverse transcribed to generate cDNA, a second strand of DNA complementary to the cDNA is generated, and a complementary RNA strand is generated) in a chamber with a small enough volume to permit detection where the amplification of target nucleic acid molecules is such that there are about 103, or fewer, RNA strands than target molecules;
and
providing conditions for detecting the amplified RNA strands by providing oligonucleotides displayed on encoded microparticles, wherein differently-encoded microparticles have different oligonucleotides attached thereto, and the oligonucleotides are elongated to generate labeled cDNA, where RNA strands complementary in whole or in part to the oligonucleotides are present.

A method of determining variable sites, including mutations and polymorphic sites (designated as normal or variant), in genomic DNA, and whether neither allele, one allele or both alleles, include the variable site, comprising:

providing conditions for amplifying genomic DNA using a polymerase chain reaction, using at least two pairs of forward-reverse primers for each variable site of interest, a first pair of forward-reverse primers designed to amplify a subsequence of the genomic DNA including the variable site and generate a pair of first amplicons including the variable site, and a second pair of forward-reverse primers wherein each member thereof includes a transcription promoter sequence and each said member of the second pair is designed to anneal to a designated target strand and be elongated, and each said member of the second pair having a nucleotide at or near its 3′ terminus which, when annealed to its designated target strand, is designed to align with the variable site and to be elongated under polymerse-chain reaction conditions only if said aligned nucleotides are complementary;
providing conditions for an in vitro transcription reaction, to reverse transcribe the amplicons into pairs of complementary RNA strands, such that the presence or the first member of the RNA strand pair identifies the normal at the variable site in the genomic DNA, and the presence or the other member of the RNA strand pair identifies the variant at the variable site in the genomic DNA;
detecting the presence of members of the RNA strand pair by capturing them to oligonucleotides, complementary in whole or in part to said members, said oligonucleotides being displayed on encoded solid supports;
and
determining the ratio of members of the RNA strand pair to each other in order to determine whether neither allele, or one allele or both alleles are present in the genomic DNA include the mutant site or a particular polymorphism, as applicable.

2 more elements

Dependencies are inferred from the claim text.

Nucleic acid amplification with integrated multiplex dectection

Life of this patent

Nucleic acid amplification with integrated multiplex dectection

Life of this patent

Abstract

Claims47