# US 10,479,989 B2 · Compositions for making random codon-mutant libraries and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,479,989 B2 - Document ID: US10479989B2 - Status: Granted - Kind: B2 - Application number: 14/898,444 - Filed: 2014-06-13 - Published: 2016-05-26 - Granted: 2019-11-19 - Earliest claimed: 2013-06-14 - Assignees: Fred Hutchinson Cancer Research Center - Applicants: Fred Hutchinson Cancer Research Center - Inventors: Jesse D. Bloom - Web page: https://patentgrep.com/patent/US10479989B2 ## CPC - C12N 15/1031: {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR} - C12N 15/102: {Mutagenizing nucleic acids} - C12N 15/67: General methods for enhancing the expression - 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 disclosure relates to compositions and methods for randomly introducing codon-mutations in a target nucleic acid molecule and, more particularly, using wild-type and triplet-randomized oligonucleotides to introduce mutations uniformly across a target nucleotide of interest in a controlled fashion and with a low rate of insertions or deletions. ## Claims ### Claim 1 A method for making a plurality of variant nucleic acid molecules, comprising (a) amplifying a reference nucleic acid molecule template with a plurality of forward mutagenic oligonucleotides to produce a first plurality of forward mutagenic fragments and separately amplifying the reference nucleic acid molecule template with a plurality of reverse mutagenic oligonucleotides to produce a first plurality of reverse mutagenic fragments, wherein the reference nucleic acid molecule template comprises a plurality of codons that encode a reference polypeptide and wherein each of the plurality of forward and reverse mutagenic oligonucleotides comprise a codon randomized at one to three nucleotides and the oligonucleotides comprise from nine to 100 nucleotides, and wherein the randomized codons correspond to all codons of the reference nucleic acid molecule template except the initiation codon; (b) purifying the first plurality of forward and reverse mutagenic fragments and mixing the purified first plurality of forward and reverse mutagenic fragments to produce a first mixed mutagenic nucleic acid molecule fragment template composition; and (c) joining the mixed nucleic acid molecule fragments of the first mixed mutagenic nucleic acid molecule fragment template composition by amplifying in a single reaction with the plurality of forward and reverse mutagenic oligonucleotides from step (a) to produce a first plurality of joined mutagenic nucleic acid molecules; thereby introducing codon variants at random locations at a controlled mutation rate across the length of the reference nucleic acid molecule template. ### Claim 2 (depends on 1) The method according to claim 1, wherein the method further comprises purifying the first plurality of joined mutagenic nucleic acid molecules and repeating steps (a) through steps (c) of claim 1 to produce a second plurality of joined mutagenic nucleic acid molecules. ### Claim 3 (depends on 1, 2) The method according to claim 2, wherein the method further comprises purifying the second plurality of joined mutagenic nucleic acid molecules and repeating steps (a) through steps (c) of claim 1 to produce a third plurality of joined mutagenic nucleic acid molecules. ### Claim 4 (depends on 1, 3) The method according to claim 3, wherein the method further comprises repeating steps (a) through steps (c) of claim 1 at least 3, 4, 5, 6, 7, 8, 9, 10 or more times by purifying the plurality of joined mutagenic nucleic acid molecules generated in the previous round of the method and performing steps (a) through steps (c). ### Claim 5 (depends on 1) The method according to claim 1, further comprising the step of amplifying the reference nucleic acid molecule template before step (a) of claim 1 with an oligonucleotide complimentary to the 5′-end of the reference nucleic acid molecule template and with an oligonucleotide complimentary to the 3′-end of the reference nucleic acid molecule template, wherein the 5′-end and 3′-end oligonucleotides comprise a sequence recognized by a restriction endonuclease to produce an end-modified reference nucleic acid molecule template; and purifying the end-modified reference nucleic acid molecule template. ### Claim 6 (depends on 1) The method according to claim 1, wherein the randomized codon of the forward and reverse mutagenic oligonucleotides is NNN, YTN, NTN, NNW, NNS, NNM, NNK, NNR, NNB, NND, NNV, NNH, BBB, DDD, VVV, HHH, NNA, NNT, NNC, NNG, NAN, NCN, NGN, ANN, TNN, CNN, GNN or any combination thereof. ### Claim 7 (depends on 1) The method according to claim 1, wherein the randomized codon of the forward and reverse mutagenic oligonucleotides is located in the middle of the oligonucleotide, near the 5′-end of the oligonucleotide, near the 3′-end of the oligonucleotide, or any combination thereof. ### Claim 8 (depends on 1) The method according to claim 1, wherein the forward mutagenic oligonucleotide, reverse mutagenic oligonucleotide, or both individually comprise from about 20 to about 50 nucleotides. ### Claim 9 (depends on 1) The method according to claim 1, wherein the codon variants at random locations across the length of the reference nucleic acid molecule template comprise a number of mutations and the number of mutations per reference nucleic acid molecule template follows an approximate Poisson distribution. ### Claim 10 (depends on 1) The method according to claim 1, wherein the codon variants at random locations across the length of the reference nucleic acid molecule template comprise mutations that occur uniformly along the length of the reference nucleic acid molecule template. ### Claim 11 (depends on 1) The method according to claim 1, wherein the codon variants at random locations across the length of the reference nucleic acid molecule template comprise a uniform base composition. ### Claim 12 (depends on 1) The method according to claim 1, wherein the reference nucleic acid molecule templates comprises multiple mutations that are not clustered. ### Claim 13 (depends on 1) The method according claim 1, wherein the number of codon variants introduced comprises from about one to about ten different mutants. ### Claim 14 (depends on 1) The method according to claim 1, wherein the method results in no more than 0.5 insertions, deletions, or a combination thereof per reference nucleic acid molecule. ### Claim 15 (depends on 1) The method according to claim 1, wherein the reference nucleic acid molecule is an animal, viral, bacterial, fungal, plant, protist, or archaebacterial gene. ### Claim 16 (depends on 15) The method according to claim 15, wherein the viral gene is an influenza gene. ### Claim 17 (depends on 16) The method according to claim 16, wherein the influenza gene is a nucleoprotein or hemagglutinin gene. ### Claim 18 (depends on 1) The method according to claim 1, wherein the forward mutagenic oligonucleotide, reverse mutagenic oligonucleotide, or both individually comprise non-coding sequence from the reference nucleic acid molecule template.