# US 10,870,879 B2 · Method for the preparation of bar-coded primer sets > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,870,879 B2 - Document ID: US10870879B2 - Status: Granted - Kind: B2 - Application number: 15/765,742 - Filed: 2016-10-05 - Published: 2019-01-03 - Granted: 2020-12-22 - Earliest claimed: 2015-10-05 - Assignees: Helmholtz Zentrum MüNchendeutsches Forschungszentrum FüR Gesundheit Und Umwelt - Applicants: Helmholtz Zentrum MüNchen—Deutsches Forschungszentrum FüR Gesundheit Und Umwelt (GmbH) - Inventors: Micha Drukker; Florian Opperer; Christian Krendl; Can Sönmezer - Web page: https://patentgrep.com/patent/US10870879B2 ## CPC - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags} - C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR} - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q 2525/155: incorporating/generating a new priming site - C12Q 2525/161: incorporating target specific and non-target specific sites - 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 - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to a method of producing a set of primers suitable for the reverse transcription and/or amplification of a plurality (N) of nucleic acid molecules of interest, wherein for each nucleic acid molecule of interest at least one primer is produced and wherein the primers carry a bar-code, the method comprising the steps of: (a)(i) combining (1) a first oligonucleotide, wherein said first oligonucleotide comprises a first bar-code nucleic acid sequence linked at its 3′ end to a first adapter nucleic acid sequence with (2) a plurality (N) of second oligonucleotides, wherein each second oligonucleotide comprises the reverse complementary sequence of a forward primer specific for a nucleic acid molecule of interest, wherein said reverse complementary sequence of the forward primer is linked at its 3′ end to the reverse complementary sequence of the first adapter nucleic acid sequence; and/or (a)(ii) combining (1) a third oligonucleotide, wherein said third oligonucleotide comprises a second bar-code nucleic acid sequence linked at its 3′ end to a second adapter nucleic acid sequence with (2) a plurality (N) of fourth oligonucleotides, wherein each fourth oligonucleotide comprises the reverse complementary sequence of a reverse primer specific for said nucleic acid molecule of interest, wherein said reverse complementary sequence of the reverse primer is linked at its 3′ end to the reverse complementary sequence of the second adapter nucleic acid sequence; wherein steps (a)(i) and (a)(ii) are carried out under conditions that enable the annealing of the first and second adapter nucleic acid sequences to the respective reverse complementary sequences thereof; (b) extending the oligonucleotides of (a)(i) and (a)(ii) by polymerase-mediated oligonucleotide synthesis; and (c) optionally, removing the second and fourth oligonucleotides. The present invention further relates to methods of producing a plurality (M) of nucleic acid amplification products of interest carrying at least one sample-specific bar-code as well as to a method for multiplex sequencing of a plurality (M) of nucleic acid amplification products of interest from a plurality (X) of samples in a single reaction chamber and identifying the individual sample from which each nucleic acid amplification product is derived. Furthermore, the present invention relates to a target-unspecific bar-code-adapter panel, its use in the methods of the invention as well as a kit comprising same. ## Claims ### Claim 1 A method of producing a plurality (M) of nucleic acid amplification products and/or reverse transcription products of interest carrying at least one sample-specific bar-code, the method comprising (i) producing a set of primers suitable for the reverse transcription and/or amplification of a plurality (N) of nucleic acid molecules of interest, wherein for each nucleic acid molecule of interest at least one primer is produced and wherein the primers carry a bar-code, the method comprising the steps of: (a)(i′) combining (1) a first oligonucleotide, wherein said first oligonucleotide comprises a first bar-code nucleic acid sequence linked at its 3′ end to a first adapter nucleic acid sequence with (2) a plurality (N) of second oligonucleotides, wherein each second oligonucleotide comprises the reverse complementary sequence of a forward primer specific for a nucleic acid molecule of interest, wherein said reverse complementary sequence of the forward primer is linked at its 3′ end to the reverse complementary sequence of the first adapter nucleic acid sequence; and/or (a)(ii′) combining (1) a third oligonucleotide, wherein said third oligonucleotide comprises a second bar-code nucleic acid sequence linked at its 3′ end to a second adapter nucleic acid sequence with (2) a plurality (N) of fourth oligonucleotides, wherein each fourth oligonucleotide comprises the reverse complementary sequence of a reverse primer specific for said nucleic acid molecule of interest, wherein said reverse complementary sequence of the reverse primer is linked at its 3′ end to the reverse complementary sequence of the second adapter nucleic acid sequence; wherein the bar-code nucleic acid sequences are at least 3 nucleotides in length, wherein the adapter nucleic acid sequences are at least 4 nucleotides in length, wherein the primer is at least 10 nucleotides in length, wherein steps (a)(i′) and (a)(ii′) are carried out under conditions that enable the annealing of the first and second adapter nucleic acid sequences to the respective reverse complementary sequences thereof; (b) extending the oligonucleotides of (a)(i′) and (a)(ii′) by polymerase-mediated oligonucleotide synthesis, wherein the polymerase is a DNA polymerase selected from the group consisting of Klenow DNA polymerase and T4 polymerase; and (c) removing the second and fourth oligonucleotides, wherein said removing is effected by exonuclease digestion; and (ii) amplifying and/or reverse transcribing a plurality (M) of nucleic acid molecules of interest from a sample using the set of primers produced in (i). ### Claim 2 (depends on 1) The method of claim 1, further comprising: (iii) producing at least a second set of primers suitable for the reverse transcription and/or amplification of a plurality (M) of nucleic acid molecules of interest, wherein for each nucleic acid molecule of interest at least one primer is produced and wherein the primers carry a bar-code, wherein said at least one second set of primers differs in its bar-code nucleic acid sequence from the first set of primers of (i); and (iv) amplifying and/or reverse transcribing said plurality (M) of nucleic acid molecules of interest from a second sample using the second set of primers produced in (iii). ### Claim 3 (depends on 2) The method of claim 2, wherein steps (iii) and (iv) are repeated for a plurality (X) of samples, wherein each set of primers produced in (iii) differs in their bar-code nucleic acid sequences from each of the other sets of primers; thereby producing from each sample a plurality (M) of nucleic acid amplification products and/or reverse transcription products of interest carrying at least one sample-specific bar-code. ### Claim 4 A method for multiplex sequencing of a plurality (M) of nucleic acid amplification products and/or reverse transcription products of interest from different samples and identifying the individual sample from which each nucleic acid amplification product and/or reverse transcription product is derived, the method comprising: (a′) producing a plurality (M) of nucleic acid amplification products and/or reverse transcription products of interest for each individual sample in accordance with the method of producing a plurality (M) of nucleic acid amplification products and/or reverse transcription products of interest carrying at least one sample-specific bar-code, the method comprising (i) producing a set of primers suitable for the reverse transcription and/or amplification of a plurality (N) of nucleic acid molecules of interest, wherein for each nucleic acid molecule of interest at least one primer is produced and wherein the primers carry a bar-code, the method comprising the steps of: (a)(i′) combining (1) a first oligonucleotide, wherein said first oligonucleotide comprises a first bar-code nucleic acid sequence linked at its 3′ end to a first adapter nucleic acid sequence with (2) a plurality (N) of second oligonucleotides, wherein each second oligonucleotide comprises the reverse complementary sequence of a forward primer specific for a nucleic acid molecule of interest, wherein said reverse complementary sequence of the forward primer is linked at its 3′ end to the reverse complementary sequence of the first adapter nucleic acid sequence; and/or (a)(ii′) combining (1) a third oligonucleotide, wherein said third oligonucleotide comprises a second bar-code nucleic acid sequence linked at its 3′ end to a second adapter nucleic acid sequence with (2) a plurality (N) of fourth oligonucleotides, wherein each fourth oligonucleotide comprises the reverse complementary sequence of a reverse primer specific for said nucleic acid molecule of interest, wherein said reverse complementary sequence of the reverse primer is linked at its 3′ end to the reverse complementary sequence of the second adapter nucleic acid sequence; wherein the bar-code nucleic acid sequences are at least 3 nucleotides in length, wherein the adapter nucleic acid sequences are at least 4 nucleotides in length, wherein the primer is at least 10 nucleotides in length, wherein steps (a)(i′) and (a)(ii′) are carried out under conditions that enable the annealing of the first and second adapter nucleic acid sequences to the respective reverse complementary sequences thereof; (b) extending the oligonucleotides of (a)(i′) and (a)(ii′) by polymerase-mediated oligonucleotide synthesis, wherein the polymerase is a DNA polymerase selected from the group consisting of Klenow DNA polymerase and T4 polymerase; and (c) removing the second and fourth oligonucleotides, wherein said removing is effected by exonuclease digestion; and (ii) amplifying and/or reverse transcribing a plurality (M) of nucleic acid molecules of interest from a sample using the set of primers produced in (i); (iii) producing at least a second set of primers suitable for the reverse transcription and/or amplification of a plurality (M) of nucleic acid molecules of interest, wherein for each nucleic acid molecule of interest at least one primer is produced and wherein the primers carry a bar-code, wherein said at least one second set of primers differs in its bar-code nucleic acid sequence from the first set of primers of (i); and (iv) amplifying and/or reverse transcribing said plurality (M) of nucleic acid molecules of interest from a second sample using the second set of primers produced in (iii); (b′) combining the nucleic acid amplification products and/or reverse transcription products produced in (a′) from all samples; (c′) sequencing the combined nucleic acid amplification products and/or reverse transcription products of (b′); and (d′) identifying the individual sample from which each nucleic acid amplification product and/or reverse transcription product is derived based on the sample-specific bar-code associated with the nucleic acid amplification product and/or reverse transcription product. ### Claim 5 (depends on 1) The method of claim 1, wherein N represents an integral number of at least 2. ### Claim 6 (depends on 1) The method of claim 1, wherein in step (i) the first and/or third oligonucleotide further comprise a 5′ protecting group.