# US RE48,732 E1 · Methods and compositions for the selection and optimization of oligonucleotide tag sequences > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US RE48,732 E1 - Document ID: USRE48732E1 - Status: Granted - Kind: E1 - Application number: 16/040,383 - Filed: 2018-07-19 - Granted: 2021-09-14 - Earliest claimed: 2011-03-10 - Assignees: Gen-Probe Incorporated - Applicants: Gen-Probe Incorporated - Inventors: Norman C. Nelson; Jijumon Chelliserry - Web page: https://patentgrep.com/patent/USRE48732E1 ## CPC - C40B 30/00: Methods of screening libraries - C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids - C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags} - C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules - G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression - G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) - C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY ## 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. ## Claims ### Claim 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 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. ### Claim 2 (depends on 1) The method according to claim 1, wherein the modification in step k) comprises systematically deleting nucleotides from the tag sequence. ### Claim 3 (depends on 1) The method according to claim 1, further comprising the steps of: (i) modifying the sequence of the tag sequence from step g); (ii) synthesizing an oligonucleotide to contain the modified tag sequence; (iii) measuring for the oligonucleotide containing a modified tag sequence 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 (iv) selecting one or more of the modified nucleic acid tag sequences used in step (iii) for use in a nucleic acid assay. ### Claim 4 (depends on 3) The method according to claim 3 1, wherein the modification in step (i) comprises systematically deleting nucleotides from the tag sequence. ### Claim 5 (depends on 1) The method according to claim 1, wherein; the performance characteristic(s) comprises one or more performance characteristic(s) selected from the group consisting of: amplification performance characteristic(s); interference with nucleic acids in the nucleic acid assay; interference with one or more oligonucleotides in the nucleic acid assay; interference with one or more target nucleic acids in the nucleic acid assay; interference with one or more amplicons in the nucleic acid assay; assay reproducibility; quantification; real-time quantification; end-point quantification; a dynamic range for detecting target nucleic acid; a limit of detection; precision of replicates; reaction kinetics; and a combination thereof. ### Claim 6 (depends on 1) The method according to claim 1, wherein: a nucleic acid sequence in the pool sub-pool is used as a tag in a the nucleic acid assay of step g) and reduces interference with a nucleic acid in the nucleic acid assay to about 95% or less compared to the amount of interference present in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in an in vitro nucleic acid assay and accelerates reaction kinetics to about 105% or more compared to the reaction kinetics in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in an in vitro nucleic acid assay and slows reaction kinetics to about 95% or less compared to the reaction kinetics in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in a nucleic acid assay and increases sensitivity for a target nucleic acid so that the amount of target nucleic acid needed to obtain a detectable signal is about 95% or less of the amount of target nucleic acid required in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in a nucleic acid assay and decreases sensitivity for a target nucleic acid so that the amount of target nucleic acid needed to obtain a detectable signal is about 105% or more of the amount of target nucleic acid required in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in a nucleic acid assay and increases replication precision by about 105% or more compared to an untagged assay. ### Claim 7 (depends on 6) The method according to claim 6 1, wherein the nucleic acid assay of step g) is an in vitro isothermal amplification assay, or the nucleic acid assay is an in vitro PCR amplification assay, or the nucleic acid assay is a sequencing assay, which generates an amplification product of the assay incorporating one of the tag sequences, which is used to identify the amplification product. ### Claim 8 (depends on 6) The method according to claim 6, wherein the tag is part of an amplification oligomer. ### Claim 9 (depends on 6) The method according to claim 6, wherein the tagged assay decreases the performance parameter by from 25% to 94%, from 50% to 94%, or from 75% to 94% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein, or wherein the tagged assay increases the performance parameter by from 105% to 150%, from 105% to 200%, or from 105% to 500% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein. ### Claim 10 (depends on 1) The method according to claim 1, wherein the tag sequence has sequences selected in step e) have a Tm that is less than or equal to 72° C.; or wherein the tag sequence has sequences have a Tm selected from the group consisting of all whole and partial numbers from 35° C. through 75° C.; or wherein the tag sequence has sequences have a primer dimer energy formation that is less than or equal to −10.0 kcal/mol; or wherein the tag sequence has sequences have a hairpin stability energy that is less than or equal to −4 kcal/mol; or wherein the 3′ region of the tag sequence sequences is less than 80% complementary to the one or more oligonucleotides in the searched database; or a combination thereof. ### Claim 11 (depends on 1) The method according to claim 1, wherein the nucleic acid assay comprises two or more target nucleic acids; or wherein the nucleic acid assay comprises a target nucleic acid combined from two or more separate samples; or wherein the nucleic acid assay comprises a target nucleic acid combined from two or more separate samples and the target nucleic acid from each separate sample includes a unique tag sequence to identify from which samples the target nucleic acid originated. ### Claim 12 (depends on 1) The method according to claim 1, wherein the database having one or more nucleic acid sequences is a collection of various nucleic acid sequences corresponding to a nucleic acid assay, a public collection of nucleic acid sequences, an aligned collection of nucleic acid sequences, the pool of nucleic acid sequences, or a combination thereof; or wherein the database having one or more nucleic acid sequences is a database containing sequence(s) that are derived from: collections of various nucleic acid sequences corresponding to a nucleic acid assay; a public collection of nucleic acid sequences; a collection of aligned sequences, the pool, or a combination thereof. ### Claim 13 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. ### Claim 14 (depends on 13) The method according to claim 13, wherein the sub-pool at step d) contains one or more of the following: nucleic acid sequences with Tm values that are within ±2 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with Tm values that are within ±5 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with Tm values that are within ±10 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±5% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±10% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±30% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents from 30% to 80%, from 40% to 70%, or from 30% to 50%; nucleic acid sequences in Table 2; and nucleic acid sequences with lengths from 5 nucleobases to 100 nucleobases. ### Claim 15 (depends on 13) The method according to claim 13, wherein the in vitro amplification reaction performed at step g) has one or more of the following performance characteristics: reduced interference between nucleic acids in the reaction when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; reaction kinetics that are accelerated by about 105% or more when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; reaction kinetics that are reduced to about 95% or less when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; increased sensitivity when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer, wherein the in vitro amplification reaction using the tagged amplification oligomer requires an amount of starting material that is about 95% or less than the minimum amount of starting material required in an untagged assay in order to obtain a detectable signal; decreased sensitivity when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer, wherein the in vitro amplification reaction using the tagged amplification oligomer requires an amount of starting material that is about 105% or more than the amount of starting material required in an untagged assay in order to obtain a detectable signal; and a replication precision that is about 105% or better when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer. ### Claim 16 (depends on 15) The method according to claim 15, wherein the tagged assay decreases the performance parameter by from 25% to 94%, from 50% to 94%, or from 75% to 94% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein; or wherein the tagged assay increases the performance parameter by from 105% to 150%, from 105% to 200%, or from 105% to 500% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein. ### Claim 17 (depends on 13) The method according to claim 13, wherein the one or more nucleic acid sequences in a database is a collection of various nucleic acid sequences corresponding to a nucleic acid assay, a public collection of nucleic acid sequences, an aligned collection of nucleic acid sequences, the pool of nucleic acid sequences, or a combination thereof; or wherein the one or more nucleic acid sequences in a database contains sequence(s) that are derived from: collections of various nucleic acid sequences corresponding to a nucleic acid assay; a public collection of nucleic acid sequences; a collection of aligned sequences, the pool, or a combination thereof. ### Claim 18 (depends on 13) The method according to claim 13, wherein the in vitro amplification assay is an isothermal amplification assay; or a multiplex amplification assay; or a PCR amplification reaction; or a combination thereof. ### Claim 19 (depends on 18) The method according to claim 18, wherein an amplicon generated in the in vitro amplification assay is used in a sequencing assay. ### Claim 20 (depends on 13) A tagged amplification oligomer containing a tag sequence obtained by the method of claim 13. ### Claim 21 (depends on 13) A multiplex in vitro amplification reaction mixture containing one or more tagged amplification oligomers, each with a tag sequence obtained by any the method of claim 13. ### Claim 22 (depends on 13) A kit for amplification of a target nucleic acid, wherein the kit contains at least one tagged amplification oligomer containing a tag sequence obtained by the method of claim 13. ### Claim 23 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. ### Claim 24 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. ### Claim 25 (depends on 24) The method of claim 24, wherein step v) measures an improved performance characteristic for at least one of the different oligonucleotides relative to a corresponding untagged oligonucleotide. ### Claim 26 (depends on 24) The method of claim 24, wherein the nucleic acid assay of step v) comprises performing an in vitro isothermal amplification assay, an in vitro PCR amplification assay, or a sequencing assay with an amplification oligomer comprising a nucleic acid tag sequence identified in step v), which generates an amplification product incorporating one of the modified tag sequences, which is used to identify the amplification product.