# US 10,865,431 B1 · Polymerase incorporation of non-standard nucleotides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,431 B1 - Document ID: US10865431B1 - Status: Granted - Kind: B1 - Application number: 15/997,339 - Filed: 2018-06-04 - Granted: 2020-12-15 - Earliest claimed: 2008-06-17 - Applicants: Steven A Benner; Zunyi Yang - Inventors: Steven A Benner; Zunyi Yang - Web page: https://patentgrep.com/patent/US10865431B1 ## CPC - C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides - C07H 21/04: with deoxyribosyl as saccharide radical - C12Q 1/6844: Nucleic acid amplification reactions - C12Q 1/6869: Methods for sequencing - C12Q 2521/101: DNA polymerase - C12Q 2525/117: incorporating modified base - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - 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) - 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 ## Abstract The disclosed invention teaches processes to create a Watson-Crick complementary copy of a preselected oligonucleotide that contain non-standard nucleotides, which form nucleobase pairs fitting the standard Watson-Crick geometry, but here said pairs are joined by hydrogen bonding patterns different from those that join standard A:T and G:C pairs. The invention further relates to polymerases that incorporate those non-standard nucleotide analogs into oligonucleotide products using the corresponding triphosphate derivatives. ## Claims ### Claim 1 A process for creating a DNA molecule that is complementary to a preselected DNA molecule, wherein said process comprises (a) contacting said preselected DNA molecule with a primer under conditions where said primer anneals to said preselected DNA molecule to form a duplex, wherein said preselected DNA molecule contains at least one non-standard nucleotide comprising a nucleobase selected from the group consisting of (b) incubating said duplex with an enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase, (c) wherein said mixture also contains nucleoside analog triphosphates that are Watson-Crick complementary to the nucleotides in said preselected DNA molecule, and (d) wherein said enzyme incorporates opposite each of the nucleotides in said preselected DNA molecule, wherein R is the point of attachment of said nucleobase to said preselected DNA molecule. ### Claim 2 (depends on 1) The process of claim 1, wherein said nucleobase is wherein R is the point of attachment of said nucleobase to said preselected DNA molecule. ### Claim 3 (depends on 1) The process of claim 1 wherein said enzyme is selected from the group consisting of Phusion, Vent and Deep Vent. ### Claim 4 (depends on 1) The process of claim 1, wherein the pH is adjusted to optimize the melting temperature of said duplex. ### Claim 5 (depends on 4) The process of claim 4, wherein said pH is between 6.5 and 7.5. ### Claim 6 (depends on 1) The process of claim 1, where multiple segments are amplified at the same time. ### Claim 7 A process for amplifying an oligonucleotide sequence, said process comprising (a) contacting said sequence in aqueous solution with a forward primer and a reverse primer, wherein said forward primer is complementary to a region at the 3′-end of said sequence, wherein the reverse primer is identical in sequence to a region at the 5′-end of said sequence, (b) incubating said solution with an enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase, (c) wherein said mixture also contains nucleoside triphosphates that are Watson-Crick complementary to the nucleotides in said oligonucleotide sequence and its complement, and (d) wherein” said enzyme incorporates a nucleotide from each of said nucleoside triphosphates opposite each of the nucleotides in said sequence and its complement, and wherein said sequence contains at least two non-standard nucleotides incorporating the nucleobase wherein R is the point of attachment of said nucleobase to said preselected DNA molecule. ### Claim 8 (depends on 7) The process of claim 7 wherein said enzyme is selected from the group consisting of Phusion, Vent and Deep Vent. ### Claim 9 (depends on 7) The process of claim 7, wherein the pH is adjusted to optimize the melting temperature of the amplicon duplex. ### Claim 10 (depends on 9) The process of claim 9, wherein said pH is between 6.5 and 7.5. ### Claim 11 (depends on 7) The process of claim 7, wherein said non-standard nucleotides are adjacent in said sequence. ### Claim 12 A process for creating a DNA molecule that is complementary to a preselected DNA molecule, wherein said process comprises (a) contacting said preselected DNA molecule in aqueous solution with a primer that is not attached to a solid support under conditions where said primer anneals to said preselected DNA molecule to form a duplex, wherein said preselected DNA molecule contains at least one non-standard nucleotide comprising a nucleobase selected from the group consisting of wherein Z is alkyl, (b) incubating said solution with an enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase, (c) wherein said mixture also contains nucleoside analog triphosphates that are Watson-Crick complementary to the nucleotides in said preselected DNA molecule, and (d) wherein said enzyme incorporates opposite each of the nucleotides in said preselected DNA molecule, wherein R is the point of attachment of said nucleobase to said preselected DNA molecule. ### Claim 13 A process for amplifying an oligonucleotide sequence, said process comprising (a) contacting said sequence in aqueous solution with a forward primer and a reverse primer, wherein said forward primer is complementary to a region at the 3′-end of said sequence, wherein the reverse primer is identical in sequence to a region at the 5′-end of said sequence (b) incubating said solution with an enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase, (c) wherein said mixture also contains nucleoside triphosphates that are Watson-Crick complementary to the nucleotides in said oligonucleotide sequence and its complement, (d) wherein said enzyme incorporates a nucleotide from each of said nucleoside triphosphates opposite each of the nucleotides in said sequence and its complement, and (e) wherein said enzyme continues to incorporate nucleotides after incorporating any non-standard nucleotides, wherein said sequence contains at least two non-standard nucleotides comprising the nucleobase wherein R is the point of attachment of said nucleobase to said preselected DNA molecule.