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

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.

FIELD OF THE INVENTION 
The present invention is drawn to a method for the efficient generation and 
selection of site-specific deoxyribonucleic acid (DNA) and ribonucleic 
acid (RNA) mutants. 
BIBLIOGRAPHY 
Full bibliographic citations to the references described hereinbelow can be 
found in the Bibliography, immediately preceding the Sequence Listing. 
DESCRIPTION OF THE PRIOR ART 
The scientific literature describes several methods for generating and 
selecting mutated nucleic acid sequences. These methods utilize different 
types of selection to increase the efficiency of mutagenesis. For 
instance, Bohnsack (1996) describes a method for site-directed mutagenesis 
which has been given the name Altered Sites II. The Altered Sites II 
mutagenesis protocol uses antibiotic resistance to select the successfully 
mutated transformants. In this protocol, a mutagenic oligonucleotide and 
an oligonucleotide that restores a frame-shift in an antibiotic resistance 
gene (referred to as the "repair" oligonucleotide) are simultaneously 
annealed to a template DNA strand which is either single-stranded DNA or 
alkaline-denatured double-stranded DNA. The complementary mutant strand is 
then synthesized using T4 DNA polymerase, and T4 DNA ligase. 
The mutant plasmids are then replicated in a mismatched repair-deficient E. 
coli mutant strain (either ES1301 or BMH 71-18). Replicated plasmids are 
then segregated by transforming into a second host, such as JM109. The key 
to this selection method is that the antibiotic resistance gene is 
initially non-functional. Activation of the gene provides the means for 
selection. However, a second antibiotic resistance gene is also required 
to effect the selection. This requires that specialized plasmids be used 
in the Altered Sites II protocol. Another notable feature of this protocol 
is that a third oligonucleotide which inactivates a second antibiotic 
resistance trait can be incorporated into the plasmid. By alternating 
repair and inactivation of the antibiotic resistance genes in the vectors, 
multiple rounds of mutagenesis can be performed without the need for 
additional subcloning steps. 
Vandeyar et al. (1988) describe an in vitro selection procedure for 
mutagenesis utilizing selective methylation of the mutant strand via 
incorporation of 5-methyl-dCTP. Site-directed mutagenesis is typically 
performed by annealing an oligonucleotide from 15-20 bases long to a 
single-stranded template. The oligonucleotide is then extended using DNA 
polymerase, and the ends of the strand are ligated. This yields a 
hetero-duplex molecule which contains a mismatched base pair at the 
mutation site. 
In the Vandeyar et al. method, after annealing of the mutagenic 
oligonucleotide, the primer strand is extended in a polymerase cocktail 
containing 5-methyl-dCTP. Methylated DNA is resistant to cleavage by the 
restriction enzyme MspI. Because only the mutant strand contains 
5-methyl-dCTP, digestion of the hetero-duplex with MspI results in 
selective cleavage of the template strand. After nicking the template 
strand by MspI digestion, treatment with exonuclease III removes the 
non-mutant strand. The remaining mutant strand is then used to transform 
competent E. coli. This method is notable in that the selection of the 
mutant strand is done by in vitro enzymatic digestion. 
Benkovic et al., U.S. Pat. No. 4,521,509, describe a method for degrading 
DNA in which a mutated strand includes 
2'-deoxyadenosine-5'-O-(1)-thiotriphosphate. The phosphorothioate 
internucleotide linkages are resistant to exonuclease III digestion. This 
allows the parent strand to be degraded, while the mutant strand remains 
intact and can be used to transform suitably competent host cells. 
Another method of site-specific DNA mutagenesis and selection is described 
by Kunkel in U.S. Pat. No. 4,873,192. In this method, uracil-substituted 
template DNA strands are prepared by propagating the initial DNA plasmid 
template in a strain of E. coli which yields uracil-substituted DNA 
templates. Following annealing and extension of a mutant strand to the 
dUTP-containing template strand, the plasmid is transformed into an E. 
coli strain which produces dUTPase, whereby the template strand is 
digested while the mutant strain remains intact. See also, European Patent 
Application No. 397 463 A3 to the Eastman Kodak Company. 
Slilaty, U.S. Pat. No. 5,071,743, describes a method of site-directed 
mutagenesis which yields a mutant formation efficiency of about 50%. This 
reference describes a process wherein a mutagenic oligonucleotide and a 
"closing" oligonucleotide are hybridized to a single-stranded linear DNA 
template. The "closing" oligonucleotide has a sequence which is 
complementary to either end of the linear DNA template and functions to 
circulize the DNA template. Upon circulization, the "closing" 
oligonucleotide forms an initiation site for polymerase-dependent 
complementary-strand synthesis. The complementary DNA strand, including 
the hybridized mutagenic and "closing" oligonucleotides is then formed 
using polymerase and ligase enzymes. The plasmids so formed are then 
transformed into a suitable host for replication. Similar to Kunkel, 
above, mutant selection is accomplished by propagating the initial DNA 
plasmid template in a strain of E. coli which yields uracil-substituted 
DNA templates. The semi-conservative replication of DNA then allows mutant 
selection based upon the decreased biological activity of 
uracil-substituted DNA in the ultimate transformed cell line. 
Another method of increasing the efficiency of site-directed mutagenesis is 
described in PCT published application Serial No. WO 93/13216. Here, a 
target DNA containing a unique restriction site is used to generate a 
mismatched mutant strand which lacks the restriction site. The resultant 
heteroduplex is then transformed into a suitable host. After several 
rounds of replication, the plasmid DNA is isolated from the transformed 
host. The isolated DNA is then treated with an enzyme which cleaves at the 
unique restriction site, thereby cleaving only plasmid derived from the 
non-mutated parental DNA strand. Suitably competent cells are then 
transformed using the digested DNA. The uncleaved mutant DNA is more 
readily transformed into the cells than the cleaved parental DNA, thereby 
increasing the yield of successfully transformed mutants. 
German Patent 4,024,187, describes a method for site-directed mutagenesis 
wherein a plasmid is cleaved at a cleavage site which is outside the 
region to be mutated. The cleavage site is then modified so that it can no 
longer be closed by a ligase. Then, with a second aliquot of the plasmid, 
the region to be mutated is excised from the plasmid and the residual 
plasmid isolated. The plasmid with the altered cleavage site and the 
plasmid with the mutation region excised are then mixed and rehybridized 
to form two partially single-stranded complementary gapped circles which 
contain the region to be mutated in single-stranded form. A mutagenic 
oligonucleotide can then be hybridized into the open gap. 
.beta.-Lactamase is a clinically important enzyme because of its 
degradative action on several widely used antibiotics. Consequently, its 
structure and function have been extensively studied. Several such studies 
are described below. 
Palzkill and Botstein (1992a) describe a structure/function analysis of 
.beta.-lactamase utilizing random mutagenesis of short stretches of the 
.beta.-lactamase gene. In this study, three to six codons of a DNA 
sequence coding .beta.-lactamase were randomly mutagenized. The mutant 
DNA's were then transformed into a suitable host and the percentage of 
random sequences which produce a functional protein determined. 
A similar study of Palzkill and Botstein (1992b) describes amino acid 
substitutions in TEM-1 .beta.-lactamase which alter the substrate 
specificity of the .beta.-lactamase. 
Several analogous studies are described in the scientific literature. For 
instance, Delaire et al. (1992) described site-directed mutagenesis at the 
codons coding for arginine 244 and methionine 69 of .beta.-lactamase. 
Venkatachalam et al. (1994) describe several broad spectrum 
.beta.-lactamase mutant enzymes which display increased catalytic action 
against both penicillins and cephalosporins. The mutants were constructed 
via cassette mutagenesis and identified by substitutions in the active 
site which altered substrate specificity. Imtiaz et al. (1994) describe 
the interaction of two specific point mutations in TM-1 .beta.-lactamase. 
In this study, arginine-244 was mutated to serine, along with an identical 
mutation at residue 164. A similar study analyzing the replacement of 
lysine-234 with arginine is described by Lenfant et al. (1991). 
SUMMARY OF THE INVENTION 
One aspect of the present invention is drawn to a method for conducting 
site-specific mutagenesis of single or double-stranded nucleic acids. The 
method includes the steps of hybridizing a first mismatched 
oligonucleotide which encodes a mutation in an antibiotic resistance gene 
to a target nucleic acid strand. Additionally, at least one other 
mismatched oligonucleotide encoding a desired mutation is hybridized to 
the target nucleic acid strand. The two hybridizations are preferably 
performed simultaneously. Then the hybridized mismatched oligonucleotides 
are extended. The resultant nucleic acid molecule is then incorporated 
into a host cell line to yield transformed cells. The transformed cells 
are then separated from non-transformed parent-type cells via a 
differential antibiotic resistance which is conferred to the transformed 
cells by the mutation encoded by the first mismatched oligonucleotide. 
Another aspect of the invention is directed to a deoxyribonucleic acid 
comprising a nucleotide base sequence as shown in SEQ. ID. NO: 1. 
A still further aspect of the present invention is a mutant gene encoding a 
mutation in a .beta.-lactamase gene product which confers increased 
.beta.-lactam antibiotic resistance to hosts transformed therewith 
comprising a nucleotide base sequence as shown in SEQ. ID. NO: 1. 
Additionally, the present invention is drawn to a kit for conducting 
site-specific mutagenesis of single or double-stranded nucleic acids by 
the above-described method. The kit includes a first receptacle containing 
a DNA molecule selected from the group consisting of SEQ. ID NO: 1 and 
SEQ. ID. NO: 2, and instructions for use of the kit. The instructions are 
a step-by-step guide to the site-directed mutagenesis protocol described 
herein. 
A distinct advantage of the present invention is that selection based upon 
the novel antibiotic resistance conferred by the first oligonucleotide 
dramatically increases the mutagenesis efficiency for the other mutated 
oligonucleotides which are incorporated into the synthesized strand. 
Desired mutants can be generated efficiently and quickly, in yields well 
in excess of 50%. 
Further aims, objects, and advantages of the mutagenesis protocol described 
and claimed herein will become apparent upon a complete examination of the 
Detailed Description, attached claims, and accompanying drawing figures.

DETAILED DESCRIPTION OF THE INVENTION 
The first step of the method of the present invention is to hybridize a 
first mismatched oligonucleotide which encodes a mutation in an antibiotic 
resistance gene to a target nucleic acid strand. It is this first 
mismatched oligonucleotide which acts as a selection tool for efficiently 
separating successfully transformed and mutated progeny from parent-type 
progeny. 
The mutation in the antibiotic resistance gene encoded by the first 
mismatched oligonucleotide confers a novel increased antibiotic resistance 
to hosts transformed with the gene. The increased antibiotic resistance 
conferred by the first oligonucleotide is not displayed in the wild-type 
of the organism transformed using the oligonucleotide. The preferred first 
mismatched oligonucleotide encodes a mutation in the enzyme gene product 
.beta.-lactamase. This mutant gene product confers increased resistance to 
.beta.-lactam antibiotics as compared to wild-type .beta.-lactamase genes. 
Preferably, the mutation encoded by the first oligonucleotide yields a 
gene product which confers increased resistance to cefotaxime, 
ceftazidime, or both. 
Additionally, at least one other mismatched oligonucleotide encoding a 
desired mutation is hybridized to the target nucleic acid strand. It is 
critical for optimum performance of the method that the desired mutation 
encoded by the at least one other oligonucleotide hybridizes to the same 
target nucleic acid strand as the first mismatched oligonucleotide. Also, 
the desired mutation encoded by the at least one other oligonucleotide 
should not interfere with the functionality of the first hybridized 
oligonucleotide. Therefore, it is preferred that the at least one other 
oligonucleotide hybridizes to a different physical location on the target 
nucleic acid strand than the first oligonucleotide. 
The desired mutation encoded by the at least one other oligonucleotide can 
be any mutation of interest to the user of the invention. So long as the 
mutation encoded by the at least one other oligonucleotide does not 
interfere with the functionality of the first oligonucleotide and can be 
hybridized to the same nucleotide strand as the first oligonucleotide, any 
mutation, without limitation, can be studied using the subject invention. 
The two hybridizations are preferably performed simultaneously, although 
they may be performed in separate steps, in any order. Hybridization can 
be accomplished by any of several methods well known to those having skill 
in the art. (Reference is made to the Examples, below, for an illustrative 
method.) 
Once the mismatched oligonucleotides have been hybridized to the same 
target nucleic acid strand, the oligonucleotides are extended. The 
extension is preferably performed using a polymerase enzyme, such as T4 
DNA polymerase, and a ligase enzyme, such as T4 DNA ligase, in a suitable 
reaction solution including dNTPs, to yield a nucleic acid molecule which 
includes the two mismatched oligonucleotides within the same strand of the 
molecule. Such extension reactions are well known in the art. (See the 
Examples, below, for a preferred method of extending the hybridized 
oligonucleotides.) 
After extension, the nucleic acids are incorporated into a suitable host. 
Preferably, the host is an E. coli. cell line. The E. coli cell line is 
preferably mismatch repair-deficient (for efficiency), although this is 
not required. The nucleic acid is more preferably transformed into E. 
coli. cell line ES1301. This cell line contains a mutation in the mutS 
gene which reduces mismatch repair in the cell, which results in a higher 
percentage of the desired mutation being formed. 
Incorporation into a host cell line to yield transformed cells can be 
accomplished by any of several well known methods, including 
transformation using calcium chloride, electroporation, and the like. For 
an exhaustive description of methods for cell transformation, see Current 
Protocols in Molecular Biology, Vol 1, Chapter 1.8, incorporated herein by 
reference for its teaching of cellular transformation. 
Successfully transformed cells will include both the mutated antibiotic 
resistance conferred by the first mismatched oligonucleotide, as well as 
the desired mutation encoded by the at least one other mismatched 
oligonucleotide. As a result, the successfully transformed cells can be 
easily separated from non-transformed parent-type cells, as well as cells 
which do not contain the mutation encoded by the first oligonucleotide, by 
the differential antibiotic resistance conferred to the transformed cells 
by the first mismatched oligonucleotide. This outcome greatly simplifies 
generating and selecting site-specific mutations. 
A preferred first oligonucleotide, SEQ. ID. NO: 1, and its reverse 
complement, SEQ. ID. NO: 2, are depicted in Table 1, below. These two 
oligonucleotides confer increased .beta.-lactam antibiotic resistance to 
E. coli. As genes incorporated into competent E. coli, genes having the 
base sequences shown in SEQ. ID. NOS. 1 and 2 provide highly useful 
selection markers for use in site-directed mutagenesis and mutant 
selection. 
The present invention also includes a kit for easily carrying out the 
above-described protocol. The kit includes at least one receptacle which 
contains a nucleic acid having a base sequence as shown in SEQ. ID. NO. 1 
or SEQ. ID. NO. 2. The kit also includes instructions on how to properly 
practice the method of the present invention. 
EXAMPLES 
The following Examples are included herein solely to provide a more clear 
and complete understanding of the invention described and claimed herein. 
The Examples do not limit the scope of the claimed invention in any manner 
whatsoever. 
Example 1 
The procedure described hereinabove was used to generate a mutation in the 
plasmid pBR322. The pBR322 plasmid contains the gene for .beta.-lactamase 
and also a resistance gene for tetracycline. The subject procedure was 
used to alter the substrate specificity of the .beta.-lactamase to confer 
resistance to ceftazidime and cefotaxime. A second mutation was introduced 
in the pBR322 plasmid to produce a frame-shift in the tetracycline 
resistance gene which inactivates the gene product. Cells containing the 
plasmid with the frame-shift in the tetracycline resistance gene do not 
grow in the presence of tetracycline. The percentage of plasmids 
containing this second mutation can easily be determined by evaluating the 
number of plasmid-containing colonies which do not grow in the presence of 
tetracycline. 
pBR322 plasmid DNA (2 .mu.g) was denatured by treatment with 0.2M NaOH and 
0.2 mM EDTA for 5 minutes at 25.degree. C. The pH was then neutralized by 
addition of 0.2M ammonium acetate pH 4.6. The DNA was then precipitated by 
addition of ethanol and isolated by centrifugation. The DNA pellet was 
dried and then resuspended in 100 .mu.l of 10 mM Tris, pH 7.9, 1 mM EDTA. 
10 .mu.l (0.2 .mu.g) of denatured template was used for each of two 
mutagenesis reactions. 
In a first reaction, the denatured template was hybridized with 
oligonucleotide SEQ. ID. NO: 1 and a tetracycline knockout (tetKO) 
oligonucleotide, SEQ. ID. NO: 5 (see Table 1, below). In a second control 
reaction, the denatured template was hybridized to the tetKO 
oligonucleotide, SEQ. ID. NO: 5, alone. Hybridization reactions were 
performed in 20 .mu.l reactions containing 20 mM Tris-HCl, pH 7.5, 10 mM 
MgCl.sub.2, and 50 mM NaCl. Template and oligonucleotides were heated to 
85.degree. C. and cooled to 25.degree. C. at approximately 1.degree. 
C./minute. Reactions were then incubated with 10 units of T4 DNA 
polymerase and 3 units of T4 DNA ligase in the presence of 13 mM Tris-HCl, 
pH 7.5, 0.5 mM dNTPs, 1 mM ATP, 2 mM DTT, 6.7 mM MgCl.sub.2, 33 mM NaCl in 
a total volume of 30 .mu.l for 90 minutes at 37.degree. C. 
Aliquots of the two reactions were transformed into E. coli. cell line 
ES1301. This cell line contains a mutation in the mutS gene which reduces 
mismatch repair in the cell, which results in a higher percentage of the 
desired mutation. Transformations were performed by incubating 1.5 .mu.l 
of the mutagenesis reactions with 100 .mu.l of ES1301 cells made competent 
by the procedure described by Hanahan (1985), incorporated herein by 
reference in its entirety. Cells and DNA were incubated on ice for 10 
minutes and then transferred to a 42.degree. C. water bath for 50 seconds. 
The transformation mixes were then placed on ice for approximately 2 
minutes and then added individually to 900 .mu.l of LB and incubated for 1 
hour at 37.degree. C. 4 ml of LB (Luria broth, also known as Lenox broth) 
was then added and the 5 ml transformations were divided into 1 ml 
aliquots. Ceftazidime was added at 0, 0.5, 2, 10, or 20 .mu.g/ml final 
concentration to the 1 ml aliquots. 
After overnight incubation at 37.degree. C., the cultures were examined for 
growth. The mutagenesis reaction containing the SEQ. ID. NO: 1 
oligonucleotide showed growth at all concentrations of ceftazidime whereas 
the transformation from the control mutagenesis showed growth only at 
concentrations of ceftazidime of 2 .mu.g/ml and below. This result 
demonstrates the ability of the SEQ. ID. NO: 1 oligonucleotide to confer 
increased resistance to ceftazidime. 
Plasmid DNA was isolated from the mutagenesis reaction containing the SEQ. 
ID. NO: 1 oligonucleotide grown in the presence of 20 .mu.g/ml of 
ceftazidime. This plasmid DNA was used to transform the E. coli cell line 
JM109. Transformants were plated on LB containing ampicillin, ceftazidime, 
or cefotaxime. Colonies were obtained in each case after incubation at 
37.degree. C. overnight. Individual colonies were then tested for 
resistance to ampicillin or tetracycline. All colonies were resistant to 
ampicillin demonstrating that the SEQ. ID. NO: 1 oligonucleotide mutation 
confers resistance to ceftazidime and cefotaxime without destroying 
resistance to ampicillin. 
Of the colonies tested, 40 of 50 from the ampicillin plate were 
tetracycline sensitive, an 80% mutagenesis rate. 50 out of 50 colonies 
from the ceftazidime and cefotaxime plates were tetracycline sensitive 
demonstrating a 100% mutagenesis rate. The slightly lower rate observed 
when the transformants are plated on ampicillin is likely due to ES1301 
cells harboring the wild-type pBR322 plasmid which is carried into the 
JM109 transformation. Plating the JM109 transformation on ceftazidime or 
cefotaxime prevents growth of the wild-type plasmid. The results from this 
experiment demonstrate a high efficiency of coupling of the selectable 
mutation in the .beta.-lactamase gene with another mutant outside of that 
gene. 
TABLE 1 
__________________________________________________________________________ 
Oligonucleotide Primers Used in the Examples. 
Oligonucleotide 
Sequence Function 
__________________________________________________________________________ 
SEQ. ID. NO: 1 
CCGCGAGACCCACCCTTGGAGGCTCCAGATTTATC 
G238S:E24OK:R241zG 
mutation in .beta.-lactamase 
SEQ. ID. NO: 2 
GATAAATCTGGAGCCTCCAAGGGTGGGTCTCGCGG 
G238S:E24OK:R241G 
mutation in .beta.-lactamase 
SEQ. ID. NO: 3 
CGCCAGGGTTCCCAGTCACGACG .beta.-galactosidase .alpha.-peptide 
frameshift 
SEQ. ID. NO: 4 
CGTCGTGACTGGGAACCCTGGCG .beta.-galactosidase .alpha.-peptide 
frameshift 
SEQ. ID. NO: 5 
GCCGGGCCTCTTGCGGGCGTCCATTCC 
tetracycline knockout 
(eteKO) sequence 
__________________________________________________________________________ 
Example 2 
The SEQ. ID. NO: 1 oligonucleotide encodes the G238S:E24OK:R241G mutations 
.beta.-lactamase. These mutations confer resistance to ceftazidime and 
cefotaxime while retaining resistance to ampicillin. In order to determine 
the best concentrations of antibiotics to select for these mutations, a 
matrix of cefotaxime and ceftazidime concentrations was evaluated for 
selection of the G238S:E24OK:R241G triple mutant. 
A matrix of antibiotic concentrations was established by performing serial 
dilutions of ceftazidime (0.05-100 .mu.g/ml) and cefotaxime (0.39-50 
.mu.g/ml) in LB containing ampicillin (100 .mu.g/ml) in a sterile 
microtiter dish. The matrix was then inoculated with a small amount of an 
ES1301 cell line transformed with either a control plasmid pKK223-3 
derivative or the pKK223-3 derivative containing the G238S:E24OK:R241G 
mutations in .beta.-lactamase. (See, for instance, Hanahan, supra.) 
Microtiter plates were incubated at 37.degree. C. overnight and cell 
growth evaluated by measuring the optical density at 630 nm. The results 
are shown in FIG. 2. An identical control matrix was inoculated with 
wild-type E. coli. The results of the control reaction are shown in FIG. 
1. 
FIG. 2 clearly shows the increased resistance to ceftazidime and cefotaxime 
conferred by the G238S:E24OK:R241G mutations. This mutation can be 
selected in ceftazidime concentrations up to 100 .mu.g/ml and cefotaxime 
concentrations up to 3.13 .mu.g/ml. In contrast, the wild-type 
.beta.-lactamase confers resistance to only up to 1.56 .mu.g/ml 
ceftazidime and 0.39 .mu.g/ml cefotaxime. (See FIG. 1.) This large 
differential between the G238S:E24OK:R241G mutations and the wild-type 
.beta.-lactamase allows for efficient selection of the G238S:E24OK:R241G 
mutations as well as secondary mutations outside the .beta.-lactamase gene 
as demonstrated in Example 1. 
Example 3 
An essential part of the procedure is that the selective oligonucleotide 
(e.g., oligonucleotide SEQ. ID. NO: 1) must be on the same strand as the 
oligonucleotide used to create the desired mutation. The following example 
demonstrates this requirement. 
The oligonucleotide SEQ. ID. NO: 2 encodes the same G238S:E24OK:R241G 
mutations to .beta.-lactamase as the oligonucleotide SEQ. ID. NO: 1 except 
its sequence is the reverse complement of the SEQ. ID. NO: 1 
oligonucleotide. Therefore, the SEQ. ID. NO: 2 oligonucleotide hybridizes 
to the opposite DNA strand than the SEQ. ID. NO: 1 oligonucleotide. 
Oligonucleotides SEQ. ID. NO: 3 and SEQ. ID. NO: 4 (see Table 1, above) 
encode frameshift mutations in the .alpha.-peptide of .beta.-galactosidase 
commonly used for blue/white screening in DNA cloning vectors. 
Introduction of this frameshift mutation into the .alpha.-peptide causes 
inactivation of the .alpha.-peptide resulting in white versus blue 
colonies when introduced into an appropriate strain such as JM109 and 
plated on media containing isopropyl-1-thio-.beta.-D-galactoside (IPTG) 
and 5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside (X-gal). This blue to 
white shift provides a convenient screen for mutagenesis efficiency. 
Oligonucleotides SEQ. ID. NO: 3 and SEQ. ID. NO: 4 hybridize to the same 
strand of the template as oligonucleotides SEQ. ID. NO: 1 and SEQ. ID. NO: 
2, respectively, in the plasmid pGEM11Zf(+). 
Mutagenesis reactions were performed as described in Example 1 using 
pGEM11Zf(+) (Promega Corporation, Madison, Wis.) as the plasmid template. 
The selective oligonucleotides (SEQ. ID. NO: 1 and SEQ. ID. NO: 2) were 
paired with the .alpha.-peptide mutagenic oligonucleotides (SEQ. ID. NO: 3 
and SEQ. ID. NO: 4) to give the following combinations: 
SEQ. ID. NO: 1/SEQ. ID. NO: 3 
SEQ. ID. NO: 1/SEQ. ID. NO: 4 
SEQ. ID. NO: 2/SEQ. ID. NO: 3 
SEQ. ID. NO: 2/SEQ. ID. NO: 4. 
Mutagenesis reactions were selected in a mixture of ampicillin (100 
.mu.g/ml), ceftazidime (25 .mu.g/ml), and cefotaxime (1.5 .mu.g/ml). 
Colonies were plated in the presence of IPTG and X-gal to allow blue/white 
screening. The results from the mutagenesis are shown in Table 2, below. 
The results show a high efficiency (&gt;70%) of mutagenesis in the 
.alpha.-peptide when the mutagenic oligonucleotide hybridizes to the same 
strand as the selective oligonucleotide (pairs SEQ. ID. NO: 1/SEQ. ID. NO: 
3 and SEQ. ID. NO: 2/SEQ. ID. NO: 4) and no detectable mutagenesis (&lt;1%) 
when selective and mutagenic oligonucleotides hybridize to opposite 
strands (pairs SEQ. ID. NO: 1/SEQ. ID. NO: 4 and SEQ. ID. NO: 2/SEQ. ID. 
NO: 3). The results show the dramatic improvement in mutagenesis 
efficiency when the alteration in substrate specificity of 
.beta.-lactamase encoded by the selective oligonucleotides is paired with 
the desired mutagenic oligonucleotide. 
TABLE 2 
______________________________________ 
Mutagenesis Results from Example 3. 
Selective Mutagenic Hybridization 
Oligonucleotide 
Oligonucleotide 
to Same Strand 
% Mutants 
______________________________________ 
SEQ. ID. NO: 1 
SEQ. ID. NO: 3 
Yes 73% 
SEQ. ID. NO: 1 
SEQ. ID. NO: 4 
No &lt;1% 
SEQ. ID. NO: 2 
SEQ. ID. NO: 3 
No &lt;1% 
SEQ. ID. NO: 2 
SEQ. ID. NO: 4 
Yes 75% 
______________________________________ 
It is understood that the present invention is not limited to the 
particular embodiments, reagents, steps, or methodologies described 
hereinabove, but embraces all such forms and modifications thereof as come 
within the scope of the attached claims. 
BIBLIOGRAPHY 
Bohnsack (1996), Methods in Molecular Biology; In vitro Mutagenesis 
Protocols; Vol. 57, M. K. Thrower, Ed., Humana Press Inc., Totowa, New 
Jersey. 
Current Protocols in Molecular Biology (1994), Vol 1, Chapter 1.8, John 
Wiley and Sons, New York, New York. 
Delaire, M., Labia, R., Samama, J. P., and Masson, J. M. (1992), The 
Journal of Biological Chemistry 267(29): 20600-20606. 
Hanahan, D. (1985), In: DNA Cloning, Vol. 1, Glover, ed., IRL Press Ltd., 
London, 109. 
Imtiaz et al. (1994), Antimicrobial Agents and Chemotherapy, (38)5: 
1134-1139. 
Lenfant et al. (1991), The Journal of Biological Chemistry, 
266(26):17187-17194. 
Palzkill and Botstein (1992a), PROTEINS: Structure, Function, and Genetics, 
14:29-44. 
Palzkill and Botstein (1992b), Journal of Bacteriology, 17:5237-5243. 
Vandeyar et al. (1988), Gene, 65:129-133. 
Venkatachalam et al. (1994), Characterization of TEM-1 .beta.-lactamase 
Mutants From Positions 238 to 241 With Increased Catalytic Efficiency For 
Ceftazidime, The Journal of Biological Chemistry, 269(38):23444-23450. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CCGCGAGACCCACCCTTGGAGGCTCCAGATTTATC35 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GATAAATCTGGAGCCTCCAAGGGTGGGTCTCGCGG35 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CGCCAGGGTTCCCAGTCACGACG23 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CGTCGTGACTGGGAACCCTGGCG23 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GCCGGGCCTCTTGCGGGCGTCCATTCC27 
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