| 主权项 |
1. A method for identifying a nucleic acid tag sequence for use in a nucleic acid assay, comprising:
a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences; b) screening the pool of nucleic acid sequences to identify two or more nucleic acid sequences having two or more performance characteristics; c) selecting one or more nucleic acid sequences, each for use as tag sequence in a nucleic acid assay; d) comparing a nucleic acid sequence or sequences from the pool of nucleic acid sequences against a database having one or more nucleic acid sequences to determine complementarity of the nucleic acid sequences from the pool of nucleic acid sequences to the database having one or more sequences, e) generating a sub-pool of nucleic acid sequences, wherein the sub-pool is a collection of nucleic acid sequences with complementarity that is less than 95% to the nucleic acid sequence(s) in the database, that is less than 90% to the nucleic acid sequence(s) in the database; that is less than 80% to the nucleic acid sequence(s) in the database, that is less than 70% to the nucleic acid sequence(s) in the database, or that is less than 50% to the nucleic acid sequence(s) in the database; f) screening the sub-pool of nucleic acid sequences for one or more performance characteristics selected from melting temperature, activity in an enzyme reaction, G-C content, nucleobase composition, length, hybridization energy, multimer formation, internal structure formation, G-quartet formation, and hairpin-stability, g) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay; h) synthesizing at least two different oligonucleotides for use in a nucleic acid assay, wherein each of the synthesized oligonucleotides has a tag sequence selected according to step g); and i) measuring for each of the different oligonucleotides synthesized in step h) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay, and optionally comparing the measurements to the measurements obtained for an untagged oligonucleotide; and j) selecting one or more of the nucleic acid tag sequences used in step i) for use in a nucleic acid assay; k) modifying the sequence of the tag sequence incorporated into an oligonucleotide from step h) to obtain a modified tag sequence for incorporation into an oligonucleotide; l) measuring for the oligonucleotide containing a modified tag sequence from step k) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay; and m) selecting one or more of the modified nucleic acid tag sequences used in step i) for use in a nucleic acid assay. |
