Mutagenesis methods and compositions

• US 5,702,931 A
• Filed: 12/28/1993
• Issued: 12/30/1997
• Est. Priority Date: 07/01/1991
• Status: Expired due to Fees

• Current Assignee: Schering Ag (Merck & Co., Inc.)
• Original Assignee: Berlex Laboratories Incorporated (Bayer AG)
• Inventors: Morser, Michael J., Vilander, Laura R., Andrews, William H.
• Primary Examiner(s): Degen, Nancy
• Application Number:
  US08/170,290
• Time in Patent Office: 1,463 Days
• Field of Search: 435/91.1, 435/91.4, 435/91.41, 435/91.42, 435/172.3, 536/24.33, 536/25.3
• US Class Current: 435/440
• CPC Class Codes: C07K 14/7455 Thrombomodulin
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/60 from viruses
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 9/6459 t-plasminogen activator (3.4.21.68), i.e. tPA
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Y 304/21069 Protein C activated (3.4.21.69)

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

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.

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

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

4. 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.

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

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

7. 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.

8. 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.

9. 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.

10. 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.

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.

12. 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.