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US 2006/0141518 A1Published application
Detection of gene expression
OwnerKai Lao, Mark Reedinventors
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Abstract
Methods for detection of nucleic acids such as a cDNA copy of an mRNA are disclosed. The methods comprise using a PCR to form a preamplification product which comprises cDNA sequence as well as primer target sequences and a detection probe sequence, which are introduced by the forward and reverse primers. In a second PCR, preamplification product is amplified using universal primers which hybridize to the primer target sequences or their complements. Amplification can be detected using a detection probe that hybridizes to the detection probe sequence or its complement.
Claims42
A method for detecting a target nucleic acid, the method comprising:
- (1) forming an initial mixture by combining components including (a) a sample suspected of comprising the target nucleic acid, (b) a polymerase, (c) a forward primer comprising a 5′ portion comprising a first primer target sequence and a 3′ portion that hybridizes to the target nucleic acid, (d) a reverse primer comprising a 5′ portion comprising a second primer target sequence and a 3′ portion that hybridizes to a complement of the target nucleic acid, and (e) a PCR reaction mix;
- wherein at least one of the forward primer and the reverse primer further comprises a detection probe sequence;
- and placing the resulting combination under conditions in which the forward primer elongates, when the target nucleic acid is a cDNA;
- (2) forming a preamplification product by subjecting the initial mixture to at least one cycle of a PCR;
- (3) forming a detection mixture comprising, in a PCR reaction mix, the preamplification product, a first universal primer that hybridizes to a complement of the first primer target sequence, and a second universal primer that hybridizes to a complement of the second primer target sequence;
- (4) amplifying any preamplification product comprised by the detection mixture;
- and
- (5) detecting amplification of any preamplification product, whereby said target nucleic acid is detected, if present in sample (1)(a), by detecting amplification of its preamplification product.
5 more elements
A method for detecting a plurality of target nucleic acids, the method comprising:
- (1) forming an initial mixture by combining components including (a) a sample suspected of comprising the plurality of target nucleic acids, (b) a polymerase, (c) a plurality of primer sets, each primer set being specific for a different target nucleic acid and each set comprising (i) a forward primer comprising a 5′ portion comprising a first primer target sequence and a 3′ portion that hybridizes to a target nucleic acid of the plurality of nucleic acids, and (ii) a reverse primer comprising a 5′ portion comprising a second primer target sequence and a 3′ portion that hybridizes to a complement of the target nucleic acid, and (d) a PCR reaction mix;
- wherein at least one of the forward primer and the reverse primer of each primer set further comprises a detection probe sequence unique for the primer set;
- and placing the resulting combination under conditions in which a forward primer elongates if hybridized to a target nucleic acid;
- (2) forming a plurality of preamplification products by subjecting the initial mixture to at least one cycle of a PCR;
- (3) forming a detection mixture comprising, in a PCR reaction mix, the plurality of preamplification products, a first universal primer that hybridizes to a complement of the first primer target-sequence, and a second universal primer that hybridizes to a complement of the second primer target sequence;
- (4) amplifying any preamplification product comprised by the detection mixture;
- and
- (5) detecting amplification of any preamplification product, whereby said target nucleic acids are detected, if present in sample (1 )(a), by detecting amplification of their preamplification products.
5 more elements
A method of detecting a target nucleic acid in a plurality of samples, the method comprising:
- (1) forming a plurality of initial mixtures, by combining, for each initial mixture, components including (a) a sample suspected of comprising the target nucleic acid, (b) a polymerase, (c) a primer set comprising (i) a forward primer comprising a 5′ portion comprising a first primer target sequence and a 3′ portion that hybridizes to the target nucleic acid, and (ii) a reverse primer comprising a 5′ portion comprising a second primer target sequence and a 3′ portion that hybridizes to a complement of the target nucleic acid, and (d) a PCR reaction mix;
- wherein at least one of the forward primer and the reverse primer further comprises a detection probe sequence unique to each probe set;
- and placing the resulting combination under conditions in which the forward primer elongates;
- (2) forming a plurality of preamplification products by subjecting each initial mixture to at least one cycle of a PCR;
- (3) forming a detection mixture comprising, in a PCR reaction mix, the preamplification products, a first universal primer that hybridizes to a complement of the first primer target sequence, and a second universal primer that hybridizes to a complement of the second primer target sequence;
- (4) amplifying any preamplification product comprised by the detection mixture;
- and
- (5) detecting amplification of any preamplification product, whereby said target nucleic acid is detected, if present in sample (1)(a), by detecting amplification of its preamplification product.
5 more elements
A method for detecting a target nucleic acid, the method comprising:
- (1) forming an initial mixture by combining components including (a) a sample suspected of comprising the target nucleic acid, (b) a polymerase, (c) a forward primer comprising a 5′ portion comprising a primer target sequence, a detection probe sequence portion, and a 3′ portion that hybridizes to the target nucleic acid, (d) a reverse primer that hybridizes to a complement of the target nucleic acid;
- and (e) a PCR reaction mix;
- and placing the resulting combination under conditions in which the forward primer elongates;
- (2) forming a preamplification product by subjecting the initial mixture to at least one cycle of a PCR under annealing conditions in which the forward primer and reverse primer each hybridize to the target nucleic acid or the complement thereof;
- (3) forming a detection mixture comprising, in a PCR reaction mix, the preamplification product, a universal primer that hybridizes to a complement of the primer target sequence, and the reverse primer;
- (4) amplifying any preamplification product comprised by the detection mixture;
- and
- (5) detecting amplification of any preamplification product, whereby said target nucleic acid is detected, if present in sample (1)(a), by detecting amplification of its preamplification product.
5 more elements
Dependencies are inferred from the claim text.