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US 9,850,529 B2Granted
Compositions and methods for nucleic acid based diagnostic assays for variable sequence targets
AssigneeBrandeis University
InventorsKenneth E. Pierce, John E. Rice, Lawrence J. Wangh
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Abstract
The present invention relates to compositions and methods for nucleic acid based diagnostic assays. In particular, the present invention provides primers for asymmetric PCR and other amplification modalities. In some embodiments, the present invention provides multiple primers for amplification of related nucleic acid targets in a single reaction.
Claims17
A method for identifying the presence of any of a plurality of related target sequences in a sample, comprising:
- (a) contacting a sample comprising a target sequence that is one of a plurality of related target sequences, wherein said target sequences differ by at least one nucleotide, with a pair of primers for amplifying one or more of said targets, wherein at least one of said primers is a thermodynamic consensus primer that is complementary to at least one of the related target sequences in the sample, and wherein the thermodynamic consensus primer comprises, at the position in which the target sequences differ, a nucleotide that minimizes the differences in melting temperature with the plurality of related target sequences rather than a consensus nucleotide which is the complement of the most common target nucleotide of the different target sequences;
- (b) performing an amplification reaction under conditions such that any of said target sequence, are amplified in a single reaction;
- and
- (c) detecting an amplification product produced in step (b).
1 more element
A method for identifying the presence of any of a plurality of related target sequences in a sample, comprising:
- (a) contacting a sample comprising a target sequence that is one of a plurality of related target sequences that comprises a position that differs in the related target sequences by at least one nucleotide with a first primer and a plurality of second primers for amplifying one of the target sequences, wherein said plurality of second primers are each complementary to a target sequence from among the plurality of related target sequences at the position that differs among the related target sequences by at least one nucleotide, or the complement thereof, and wherein one primer of said plurality of second primers is present at a concentration that is at least 5-fold greater than the concentration of each of the other primers in said plurality of second primers;
- (b) performing an amplification reaction under conditions such that said target sequence is amplified by said first primer and said plurality of second primers in a single reaction;
- and
- (c) detecting an amplification product produced in step (b).
1 more element
Dependencies are inferred from the claim text.