Back to results
US RE48,732 E1Granted
Methods and compositions for the selection and optimization of oligonucleotide tag sequences
AssigneeGen-Probe Incorporated
InventorsNorman C. Nelson, Jijumon Chelliserry
Life of this patent
Estimating the term…
Abstract
Methods for selecting tag-oligonucleotide sequences for use in an in vitro nucleic acid assay. The selected tag sequences are useful for nucleic acid assay wherein interference between the nucleic acid sequences is the assay is to be controlled. Selected tag sequences are incorporated into nucleic acid assay to improve the performance of and/or minimize any interference between nucleic acids in the assay compared to untagged assays.
Claims26
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 b) 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 c) 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 d) 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 e) selecting one or more at least one nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay;
- h f) 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 e);
- and
- i g) measuring for each of the different oligonucleotides synthesized in step h f) 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 without the tag sequence, wherein the tags of the different oligonucleotides do not serve as binding sites for amplification oligomers in the nucleotide acid assay;
- and
- j h) selecting one or more of the nucleic acid tag sequences used in step i g)for use in a nucleic acid assay;
- k i) modifying the sequence of the a tag sequence incorporated into an oligonucleotide from step h) to obtain a modified tag sequence for incorporation into an oligonucleotide;
- l j) measuring for the oligonucleotide containing a modified tag sequence from step k i) 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 k) selecting one or more of the modified nucleic acid tag sequences used in step i j) for use in a nucleic acid assay.
16 more elements
A method for identifying nucleic acid tag sequences for use in an in vitro nucleic acid amplification assay, comprising the steps of:
- a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences from Table 1;
- b) screening the pool of nucleic acid sequences against a database containing one or more nucleic acid sequences to identify percent complementarity between nucleic acid sequences in the pool and nucleic acid sequences in the database;
- c) screening the pool of nucleic acid sequences to determine a performance characteristic selected from the group consisting of:
- G-C content, nucleobase composition, length, multimer formation, primer-dimer formation, Tm, hairpin stabilization energy, self dimer stabilization energy, internal structure formation, G-quartet formation, hybridization energy, activity in an enzyme reaction, and combinations thereof
- d) generating a sub-pool of nucleic acid sequences from the results obtained in step b), step c) or steps b) and c);
- e) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay;
- f) synthesizing an amplification oligomer containing a tag sequence selected at step e);
- and
- g) performing an in vitro nucleic acid amplification reaction using the amplification oligomer.
6 more elements
A collection of nucleic acid sequences useful as tag sequences for use in a nucleic acid assay, wherein the collection contains at least two sequences in Table 1 or the collection is Table 1, Table 2 or Table 3.
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, each having two or more performance characteristics;
- and
- c) selecting one or more nucleic acid sequences with the two or more performance characteristics identified in step b), each for use as tag sequence in a nucleic acid assay;
- wherein step b) comprises:
- i) 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;
- ii) generating a sub-pool of the nucleic acid sequences of step i), 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;
- iii) 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;
- iv) 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 iii);
- and
- v) measuring for each of the different oligonucleotides synthesized in step iv) 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 comparing the measurements to measurements obtained for an oligonucleotide lacking the tag, wherein the tags of the different oligonucleotides do not serve as binding sites for amplification oligomers in the nucleic acid assay, thereby identifying one or more nucleic acid tags sequences for use in a nucleic acid assay.
9 more elements
Dependencies are inferred from the claim text.