Site-specific mutagenesis and mutant selection utilizing antibiotic-resistant markers encoding gene products having altered substrate specificity
First Claim
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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.
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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.
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Citations
14 Claims
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1. A method for conducting site-specific mutagenesis of single or double-stranded nucleic acids comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. An isolated deoxyribonucleic acid consisting of SEQ. ID. NO:
- 1.
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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.
- -lactamase gene product which confers increased β
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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.
- 1 and SEQ. ID. NO;
Specification