Site-specific mutagenesis and mutant selection utilizing antibiotic-resistant markers encoding gene products having altered substrate specificity

• US 5,780,270 A
• Filed: 07/17/1996
• Issued: 07/14/1998
• Est. Priority Date: 07/17/1996
• Status: Expired due to Fees

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Lesley, Scott A.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/682,073
• Time in Patent Office: 727 Days
• Field of Search: 435/6, 435/810, 435/91.2, 435/91.1, 435/320.1, 536/23.1, 536/22.1
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Methods, kits, and reagents for conducting site-specific mutagenesis of single or double-stranded nucleic acids which utilizes novel antibiotic resistance conferred by a mutated antibiotic resistance gene for efficient mutant selection are described.

Claims

1. A method for conducting site-specific mutagenesis of single or double-stranded nucleic acids comprising:

(a) hybridizing a first mismatched oligonucleotide which encodes a mutation in an antibiotic resistance gene to a target nucleic acid strand;

(b) hybridizing at least one other mismatched oligonucleotide encoding a desired mutation to the target nucleic acid strand;

then

(c) extending the hybridized mismatched oligonucleotides to yield an extended nucleic acid molecule;

then

(d) incorporating the extended nucleic acid molecule of step (c) into a host cell line to yield transformed cells; and

then

(e) separating the transformed cells from step (d) from non-transformed parent cells and cells lacking the mutation encoded by the first mismatched oligonucleotide via a differential antibiotic resistance conferred by the first mismatched oligonucleotide, whereby transformed cells containing the desired mutation are selected.

2. The method according to claim 1, wherein in step (a) the target nucleic acid strand is a deoxyribonucleic acid.

3. The method according to claim 1, wherein in step (a) the target nucleic acid strand is a ribonucleic acid.

4. The method according to claim 1, wherein in step (a) a first mismatched oligonucleotide encoding a mutation in β

-lactamase is hybridized to the target nucleic acid.

5. The method according to claim 1, wherein in step (a) SEQ. ID. NO. 1 is hybridized to the target nucleic acid.

6. The method according to claim 1, wherein in step (a) SEQ. ID. NO. 2 is hybridized to the target nucleic acid.

7. The method according to claim 1, wherein in step (d) the extended nucleic acid molecule is incorporated into an E. coli cell.

8. The method according to claim 7, wherein in step (d) the extended nucleic acid molecule is incorporated into a mismatch repair-deficient E. coli cell.

9. The method according to claim 7, wherein step (d) the extended nucleic acid molecule is incorporated into an E. coli strain ES1301 cell.

10. The method according to claim 1, wherein in step (e), the transformed cells are separated via antibiotic resistance to cefotaxime.

11. The method according to claim 1, wherein in step (e), the transformed cells are separated via antibiotic resistance to ceftazidime.

12. An isolated deoxyribonucleic acid consisting of SEQ. ID. NO:

1.

13. An isolated mutant gene encoding a β

-lactamase gene product which confers increased β

-lactam antibiotic resistance to hosts transformed therewith comprising a nucleotide base sequence as shown in SEQ. ID. NO;

1.

14. A kit for conducting site-specific mutagenesis of single or double-stranded nucleic acids comprising a receptacle containing a DNA molecule selected from the group consisting of SEQ. ID NO:

1 and SEQ. ID. NO;

2.