# US 5,702,931 A · Mutagenesis methods and compositions

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- Number: US 5,702,931 A
- Document ID: US5702931A
- Status: Granted
- Kind: A
- Application number: 08/170,290
- Filed: 1993-12-28
- Granted: 1997-12-30
- Earliest claimed: 1991-07-01
- Assignees: Berlex Laboratories, Inc.
- Inventors: William H. Andrews; Michael J. Morser; Laura R. Vilander
- Web page: https://patentgrep.com/patent/US5702931A

## CPC

- C12N 15/86: Viral vectors
- C07K 14/7455: {Thrombomodulin}
- C12N 9/6459: {t-plasminogen activator (3.4.21.68), i.e. tPA}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/85: for animal cells
- C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- C12Q 1/6858: Allele-specific amplification
- C12Y 304/21069: Protein C activated (3.4.21.69)
- C12N 2710/14143: viral genome or elements thereof as genetic vector
- C12N 2800/108: episomal vectors
- C12N 2830/00: Vector systems having a special element relevant for transcription
- C12N 2830/002: inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
- C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA
- C12N 2830/60: from viruses
- C12N 2830/85: mammalian
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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
- C12Y: ENZYMES

## Abstract

Methods and reagents for oligonucleotide-directed mutagenesis of a target nucleic acid are provided. In these methods a mutagenic oligonucleotide introduces a desired mutation at one site and, at a second site, introduces or eliminates a restriction site, allowing one to screen for the desired mutation by restriction analysis. Also provided are vectors and kits for performing such mutagenesis methods.

## Claims

### Claim 1

A method for introducing a plurality of mutations to a nucleic acid construct comprising a target sequence, a first marker sequence encoding a first protein having an inoperable first marker activity, and a second marker sequence encoding a second protein having an operable second marker activity, said method comprising the steps of:
(a) annealing to a single-stranded form of said nucleic acid construct
a first oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said first oligonucleotide is selected so as to change a nucleotide at a first position of said nucleic acid construct where said first position is within said target sequence, and to introduce or remove a first restriction site at a second position of said nucleic acid construct;
a second oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said second oligonucleotide is selected so as to change one or more nucleotides in said first marker sequence to produce a mutated first marker sequence in a manner which results in an operable first marker activity; and
a third oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said third oligonucleotide is selected so as to change one or more nucleotides in said second marker sequence to produce a mutated second marker sequence in a manner which results in an inoperable second marker activity, thereby forming a primary annealed product;
(b) transforming a host cell with said primary annealed product;
(c) screening or selecting progeny of transformed host cells having said operable first marker activity;
(d) identifying progeny screened or selected and containing a mutated nucleic acid construct containing a mutated target sequence and having said introduced or removed first restriction site;
(e) annealing to a single-stranded form of said mutated nucleic acid construct
a fourth oligonucleotide having a sequence substantially complementary to a portion of said mutated nucleic acid construct, wherein the sequence of said fourth oligonucleotide is selected so as to change a nucleotide at a first position of said mutated nucleic acid construct where said first position is within said mutated target sequence, and to introduce or remove a second restriction site at a second position of said mutated nucleic acid construct;
a fifth oligonucleotide having a sequence substantially complementary to a portion of said mutated nucleic acid construct, wherein the sequence of said fifth oligonucleotide is selected so as to restore said first marker sequence in a manner which results in said inoperable first marker activity; and
a sixth oligonucleotide having a sequence substantially complementary to a portion of said mutated nucleic acid construct, wherein the sequence of said sixth oligonucleotide is selected so as to restore said second marker sequence in a manner which results in said operable second marker activity, thereby forming a secondary annealed product;
(f) transforming a host cell with said secondary annealed product;
(g) screening or selecting progeny of transformed host cells having said operable second marker activity; and
(h) identifying progeny screened or selected and containing a further mutated nucleic acid construct having said introduced or removed second restriction site.

### Claim 2 (depends on 1)

The method of claim 1 wherein said target sequence encodes a polypeptide.

### Claim 3 (depends on 2)

The method of claim 2 wherein said target sequence encoding a polypeptide is operably linked to a promoter.

### Claim 4 (depends on 1)

The method of claim 1 wherein said target sequence encodes a polypeptide and the sequences of both said first and fourth oligonucleotides are each selected so as to change one or more nucleotides in said target sequence in a manner which results in a change in an amino acid of said polypeptide encoded by said target sequence, and to introduce or remove a restriction site from said nucleic acid construct in a manner which does not result in a change in an amino acid of said polypeptide.

### Claim 5 (depends on 1)

The method of claim 1 further comprising the step of extending said first, second, and third oligonucleotides with a DNA polymerase.

### Claim 6 (depends on 1)

The method of claim 1 further comprising the step of adding a DNA ligase.

### Claim 7 (depends on 1)

The method of claim 1 wherein said nucleic acid construct comprises:
(a) A ColE1 origin of replication;
(b) An M13 or f1 phage origin of replication; and
(c) A polylinker situated within a lacZ gene.

### Claim 8 (depends on 1)

The method of claim 1 wherein said target sequence encodes a polypeptide and said target sequence is operably linked to a eukaryotic promoter and said nucleic acid construct further comprises a marker for selecting eukaryotic cells into which said nucleic acid construct is introduced.

### Claim 9 (depends on 1)

The method of claim 1 wherein said nucleic acid construct is pBBS82, pBBS83, pBBS92, pBBS95, pBBS96, pBBS97, pBBS98, pBBS99, pBBS100, pBBS101, pBBS104, pBBS121, pBBS122, pBBS124, pBBS125, pBBS135, pBBS141, or pBBS145.

### Claim 10 (depends on 1)

The method of claim 1 wherein said first oligonucleotide in step (a) is selected so as to change one or more nucleotides at a plurality of positions within said target sequence, and wherein said fourth oligonucleotide in step(e) is selected so as to change one or more nucleotides at a plurality of positions within said mutated target sequence.

### Claim 11

A kit for introducing one or more mutations to a nucleic acid construct comprising:
(a) said nucleic acid construct comprising a ColE1 origin of replication, a polylinker within a lacZ gene for inserting a target sequence, a first marker sequence encoding a first protein having an inoperable first marker activity, and a second marker sequence encoding a second protein having an operable second marker activity;
(b) a first oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said first oligonucleotide is selected so as to change one or more nucleotides in said first marker sequence to produce a mutated first marker sequence in a manner which results in an operable first marker activity;
(c) a second oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said second oligonucleotide is selected so as to change one or more nucleotides in said second marker sequence to produce a mutated second marker sequence which results in an inoperable second marker activity;
(d) a third oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said third oligonucleotide is selected so as to restore said first marker sequence in a manner which results in said inoperable first marker activity;
(e) a fourth oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said fourth oligonucleotide is selected so as to restore said second marker sequence in a manner which results in said operable second marker activity;
(f) a DNA polymerase;
(g) deoxyribonucleotides;
(h) a DNA ligase: and
(i) buffers suitable for activity of said DNA polymerase and said DNA ligase.

### Claim 12 (depends on 11)

The kit of claim 11 wherein said nucleic acid construct is pBBS82, pBBS83, pBBS92, pBBS95, pBBS96, pBBS97, pBBS98, pBBS99, pBBS100, pBBS101, pBBS104, pBBS121, pBBS122, pBBS124, pBBS125, pBBS135, pBBS141, or pBBS145.