PROTOCOL

PCR-based gene targeting in Candida albicans

Andrea Walther & Jürgen Wendland
Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark. Correspondence should be addressed to J.W. (jww@crc.dk).

Published online 14 August 2008; doi:10.1038/nprot.2008.137

PCR-based gene-targeting approaches have increased the speed of gene function analyses in ascomycetous fungi, for example, in the
diploid human fungal pathogen Candida albicans. Here we describe a protocol that utilizes Rapid-PCR to amplify all cassettes available
with the previously reported pFA modules. With this protocol, sufficient quantities of any cassette for use in C. albicans
transformation experiments can be reliably generated in 25–50 min using either of the two alternative optimized amplification
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

conditions; cassette amplification by standard PCR methods typically takes 3–4 h and is likely to require optimization of
amplification conditions for each cassette. Transformants that appear 2–4 d after transformation can be rapidly identified using
Rapid-PCR on whole cells, eliminating the need for genomic DNA extraction. In total, less than a week is required for the deletion of
one allele in C. albicans. Repeating this procedure will result in the generation of homozygous mutant strains.

INTRODUCTION
Gene targeting in fungi
Gene targeting describes DNA-mediated transformation in which
a gene of interest is deleted by homologous recombination
and replaced by a selectable marker. Constructs for gene targeting
can be generated by cloning genomic DNA flanking the target locus
upstream and downstream of a marker gene. Upon the use of
suitable restriction enzymes, these disruption cassettes can be
liberated and used for transformation. This method requires
detailed knowledge of the target locus to obtain the flanking
DNA regions, which can be several hundred base pairs up to several
kilo base pairs in size. The length of the flanking regions required
for efficient homologous recombination may be species specific1. In
ascomycetous fungi, such as Saccharomyces cerevisiae, these flank-
ing regions can be in the range of 40–100 bp. This allows the
addition of flanks to a selectable marker gene using PCR.
PCR-based gene targeting in fungi
PCR-based gene targeting methods were first introduced in
S. cerevisiae by Baudin et al.2 in 1993. Many improvements have
been published in subsequent years, and this method has been
applied as a standard procedure in a variety of ascomycetous fungi
such as Ashbya gossypii, C. albicans and Schizosaccharomyces pombe,
as well as in bacteria3–10. This method has proven useful for the
deletion of the complete gene set of S. cerevisiae11,12.
The major advantage of this procedure is that primers used to
amplify selectable marker genes provide the flanking homology
that is used to direct homologous recombination at the target locus.
Thus, the resulting PCR-products can directly be used to transform
competent cells, thereby bypassing the need to clone disruption
cassettes. Furthermore, the use of short flanking homology regions
requires only minimal knowledge of the target locus sequence,
which often is available from Expressed Sequence Tags (ESTs).
However, certain limitations apply to this method. First, the
transformation efficiency of most fungal species is much lower than
that observed in S. cerevisiae13. More importantly, however, if the
degree of nonhomologous recombination is much greater than that
of homologous recombination, then longer flanking homology
regions are advisable. In fungal species only distantly related to
S. cerevisiae, nonhomologous integration may generate a large
number of false-positive transformants. This can be circumvented
by either using mutants in which the nonhomologous end-
joining pathway has been deleted—such as in Ku70 mutants of
an increasing number of fungi, including Neurospora crassa and
Sordaria macrospora—or using split-marker fragments for
transformation14–17. For split-marker transformations, two PCR
products are generated that carry sufficient flanking target homol-
ogy regions and include overlapping but nonfunctional parts of a
single selectable marker gene. The composite gene will be fused into
a functional selectable marker in vivo by homologous recombina-
tion that results also in correct insertion of the cassette at the target
locus. This tool is generally applicable and has, for example, been
used in Aspergillus nidulans and Cryptococcus neoformans10,17.
Tools for functional analysis of C. albicans
Candida albicans is a diploid organism, which prevents the use of
traditional mutagenic approaches, such as UV light or chemical
mutagenesis. Due to the absence of a haplophase, both alleles of a
gene need to be inactivated to be able to study recessive mutant
phenotypes. Several methods have been developed to exploit
homologous recombination to achieve targeted gene deletions in
C. albicans.
URA blaster. This technique uses cloned cassettes, which replace
the target gene with an URA3 selection marker18. This will generate
a heterozygous C. albicans mutant. As the URA3 gene in these
cassettes is flanked by direct repeats, recombination through these
repeats will eliminate the URA3 gene. The random occurrence of
such an event can be screened for as ura- cells are resistant to
5¢-fluoro-orotic acid, a compound that is converted into toxic
5¢-fluoro uridin monophosphate by Ura3. This selection step will
‘recycle’ the URA3 marker, allowing it to be used again to generate a
homozygous deletion mutant. Initially, this method was the
most generally applied tool for making C. albicans knockouts.
However, two problems arise when URA3 is used as a selectable
marker. First, insufficient expression of URA3 may cause defects in
cell-wall composition and lead to altered virulence in mouse
models of systemic infection that are unlinked with the gene
under investigation19,20. Secondly, the use of 5¢-FOA for counter-
selection may induce chromosomal alterations, producing
additional mutations21.

1414 | VOL.3 NO.9 | 2008 | NATURE PROTOCOLS


SAT1 flipper. This method overcomes both the problematic
features of the ‘URA blaster’ system22. A dominant selectable
marker gene conferring resistance to the antibiotic nourseothricin
is used, which does not have the adverse effects associated with
URA3. Recycling of the marker can be achieved using FLP recom-
binase, which recognizes terminal FLP target sequences. The SAT1
flipper technique has the advantage of being quite versatile and can
also be used on clinical wild-type isolates that do not carry any
auxotrophies. On the other hand, it requires several cloning steps.
Fusion PCR. This method is used to add long flanking homology
regions to selectable marker genes that afterward can be directly
used for transformation23. By using heterologous marker genes
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols
from Candida dubliniensis (CdHIS1) and Candida maltosa
(CmLEU2), integration at the C. albicans HIS1 and LEU2 loci is
prevented. Use of these markers in combination with novel
C. albicans strains has generated a very useful set of tools for the
analysis of C. albicans genes.
PCR-based gene targeting. This method does not use any cloning
steps to obtain sufficient quantities of transformation cassettes. The
usefulness of PCR-based approaches relies on the generation of
versatile modules that can be used with a limited primer set harboring
the flanking homology regions (60–100 bp) to obtain cassettes for a
large variety of functional analyses. Alterations such as precise
complete ORF deletions; promoter-swap experiments; placing
genes under the control of regulatable or very strong promoters;
and tagging of genes with epitopes or green fluorescent protein (GFP)
at either the 5¢- or 3¢-end are standard applications. This arsenal of
possible precise alterations is achievable due to the availability of
plasmid modules that serve as templates for the PCR24–28. The
modular setup of these vectors, for example, in the pFA series used
for C. albicans gene alterations, allows the replacement of particular
segments, for example, in the case new selectable marker genes or
novel promoters become available. Correct integration of the target-
ing cassettes can be verified by diagnostic PCR. Further analysis of
transformants by genomic Southern blots can be carried out to
exclude the ectopic integration of a cassette elsewhere in the genome.
Rapid-PCR-based gene targeting
Here we describe the PCR-based gene targeting method that we
have been improving since 2003 for the generation of transforma-
tion cassettes for gene function analysis in C. albicans26,28–31 (see
Supplementary Table 1 online). Although we concentrate here on
Rapid-PCR, standard PCR can also be used, and a protocol for this
can be found in Box 1.
Our Rapid-PCR protocol allows the amplification of the whole
array of pFA modules in less than an hour using one of two
alternative amplification conditions (see Supplementary Table 2
online for oligonucleotides used in conjunction with the pFA
modules). After transformation, the primary C. albicans transfor-
mants can be analyzed using a specific diagnostic Rapid-PCR
protocol. This technique allows high-throughput generation of
transformation cassettes and also overcomes limitations in the
verification of large numbers of transformants. The method can
also be applied to other fungal and bacterial systems.
Furthermore, the fast turnaround times of Rapid-PCR makes the
use of this method very attractive for all aspects of diagnostic
PCR, which ranges from the verification of correct integration of
PROTOCOL
transformation cassettes as outlined in this protocol to the verifica-
tion of cloning projects. Use of Rapid-PCR on whole Escherichia
coli cells may eventually supersede miniprep plasmid DNA isolation
to check for correct cloning of an insert, as it is much faster.
Furthermore, due to its increased sensitivity, Rapid-PCR could be
routinely implemented in clinical applications to monitor the
presence of known pathogens in patient samples.
Experimental design
Principle of Rapid-PCR. PCRs are generally carried out using a
heat-stable Taq-DNA polymerase and PCR machines that hold up to
96 tubes32. The speed of a PCR largely depends on the times required
for denaturing of the double-stranded DNA, primer annealing and
primer extension, adding, of course, the time necessary to heat or cool
the whole system. Thus, amplification in a standard reaction using a
50 ml volume will take approximately 3 h and possibly longer for
more complex protocols (see Box 1). Rapid-PCR defines reaction
settings in which 30 cycles can be obtained in less than 1 h. To achieve
this, much faster temperature adjustments are necessary, and this is
enabled by using smaller reaction volumes and special ultra-thin
microtiter-reaction plates in a SpeedCycler (Analytik Jena AG).
Shorter annealing times actually lead to a higher specificity of
primer-to-template annealing and thus more specific PCR amplifica-
tion. We use a standard Taq-polymerase for amplification of the
cassettes. The use of proofreading polymerases can be implemented
to avoid the inclusion of sequence errors, for example, in GFP fusions.
Gene-targeting cassettes. Several laboratories have taken the
effort to generate cassettes for PCR-based gene targeting in
C. albicans6,23–26,28. These include cassettes for gene deletion,
promoter exchange and GFP tagging (Fig. 1). Depending on the
strain to be used in transformation, a specific set of selectable
markers are available (see Table 1). Thus, for clinical strains, the
dominant antibiotic resistance gene SAT1 can be used, preferably as
a recyclable marker; for the BWP17 strain, the ARG4, HIS1, SAT1
and URA3 marker genes are available; and for SN148, this selection
has been expanded to five marker genes: ARG4, HIS1, LEU2, URA3
and SAT1. The use of heterologous selectable markers reduces the
potential of integration of the marker in the endogenous locus of
the transformed strain; for example, C. dubliniensis ARG4 and HIS1
and the C. maltosa LEU2 genes have been successfully used as
marker genes in C. albicans. Another aspect worth consideration is
the length of the amplified cassette. Shorter cassettes can be
amplified more easily and often with higher yields.
Primer design. Diagnostic primers used for the verification of
correct integration are o30 bases and can be synthesized at larger
scales (e.g., 0.2 mmol), as they are stable when frozen. As a storage
buffer, we recommend 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA.
Annealing temperatures of the primers are generally around 65 1C,
with a GC content close to 50%. Primers carrying a flanking
homology region are composed of the region that anneals to the
cassette at their 3¢-end (also o30 bases) and the homology region at
their 5¢-end. We have set these annealing regions to a specific region
within the pFA module that was found suitable for module construc-
tion. We also experimented with deviating regions, which produced
comparable results. For the amplification of marker–promoter mod-
ules, we use promoter-specific S2-annealing regions. This is done to
generate ATG fusion with the respective promoter and the target gene.
NATURE PROTOCOLS | VOL.3 NO.9 | 2008 | 1415
 PROTOCOL

BOX 1 | PROTOCOL FOR GENE TARGETING USING STANDARD PCR

If equipment is not available for Rapid-PCR, standard PCR can be used, but the protocol will take longer to complete.
1. Set up four 50 ml of PCR mixture in 0.2 ml of microfuge tubes as indicated below.
m CRITICAL STEP Remember to include positive and negative control reactions appropriate to the experiment, using suitable primers and
templates (see INTRODUCTION).

Reagent Amount (ll) Final concentration

pFA-plasmid template (10 ng ml
1) 5 1 ng ml
1

10 Taq polymerase buffer (including 15 mM MgCl2) 5 1
dNTPs (2 mM stock) 5 0.2 mM
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

S1-primer (2 pmol ml
1) 5 0.2 mM

S2-primer (2 pmol ml
1) 5 0.2 mM
Taq-DNA polymerase (5 U ml
1) 0.5 0.5 U

ddH2O 25

2. Transfer tubes to the PCR machine. To amplify standard modules, follow option A. To amplify composite modules, follow option B.
(A) Amplification of standard modules
(i) Program the thermocycler as outlined in the table below and run the PCR. The duration of the PCR with a polymerization step of 120 s at
72 1C is 3.5 h and with 180 s at 72 1C is 4 h.

Cycle Denaturation Annealing Polymerization

1 300 s at 94 1C — —
2–41 60 s at 94 1C 60 s at 52 1C 120–180 s at 72 1C
42 — — 360 s at 72 1C; Storage at 12 1C

(B) Amplification of composite pFA modules

(i) Program the thermocycler as outlined in the table below and run the PCR. The duration of the PCR with a polymerization step of 120 s at
72 1C is 3.5 h and with 300 s at 72 1C is 5.5 h

Cycle Denaturation Annealing Polymerization

1 300 s at 94 1C — —
2–11 60 s at 94 1C 60 s at 45 1C 120–300 s at 72 1C
12–41 60 s at 94 1C 60 s at 55 1C 120–300 s at 72 1C
42 — — 360 s at 72 1C; Storage at 12 1C

3. Pool the four PCR mixtures for a single transformation and verify the amount of PCR product by gel electrophoresis using 8 ml of the product.
4. Carry out transformation of C. albicans as described in Step 5 of the main PROCEDURE.
5. Prepare cells for colony PCR as described in Step 6 of the main PROCEDURE.
6. Prepare PCR mixtures as outlined in step 1 of this procedure using 5 ml of the cell suspension as template DNA.
7. Run a standard PCR using option B of step 2 in this procedure.
8. Check the PCR products on an agarose gel. For further evaluation of the transformants, refer to Step 10 of the main PROCEDURE.
 TIMING
Step 1: 15 min
Step 2: 3.5–5.5 h, depending on the PCR running conditions
Step 3: 30 min
Step 4: 2–4 d
Step 5: up to 2 h
Step 6: 15 min
Step 7: 3.5–5.5 h, depending on the PCR running conditions
Step 8: 30 min

The length of the flanking homology region may vary. Some studies
report that as little as 43 bases of homology to the target locus can be
used33. In our studies, we favor the use of longer homology regions of
100 bases, which produces correct transformants much more con-
sistently (Supplementary Table 2).
Controls. Several parameters may influence the fidelity of
the PCR. Long primers for the amplification of cassettes may
reduce the product yield. This can be controlled by using
short standard primers that contain only the homology region.
The amount of template can be optimized within 1–10 ng ml
1,

1416 | VOL.3 NO.9 | 2008 | NATURE PROTOCOLS

 PROTOCOL

Figure 1 | Amplification of pFA cassettes Marker Marker Promoter Marker p GFP GFP Marker
using the SpeedCycler. The repertoire of pFA CaHIS1- CaARG4- CaARG4- CdHIS1- CdHIS1-
GFP- GFP-
CdHIS1 CmLEU2 CaURA3 CaSAT1 CaARG4 M M AgTEF1p- CaMAL2p-
modules allows the amplification of cassettes AgTEF1p CaMAL2p CaMET3p GFP GFP
CmLEU2 CaARG4

with a minimal primer set. These cassettes 1,4 1,6 1,6 2,2 2,2 2,0 2,7 3,6 2,5 2,9 2,6 3,2
can be used for gene disruption, which requires
only the amplification of a marker gene; for
promoter exchange using marker–promoter
cassettes; and for GFP tagging either at the 5¢-
or the 3¢-end of a gene. Cassettes up to 2.5 kb
were amplified using the short protocol (30-s
polymerization), whereas larger cassettes were
obtained using the longer protocol (45-s
polymerization). Fragments were separated
on an ethidium bromide-stained 0.8%
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

(wt/vol) agarose gel and imaged using a

Pharmacia Biotach ImageMaster gel
documentation unit. Size standard M is
l-DNA-cleaved with PstI.

and linearization of the plasmid DNA may also increase the yield
of PCR products. Due to size differences of the cassettes, yields may
be higher when using shorter cassettes as template (e.g., derived
from CdHIS1).
Colony PCRs should be set up to include positive controls
performed on whole cells. We use internal gene fragments amplified
with I1 and I2 primers when generating heterozygous mutants.
Primers not related to the gene of interest that result in a
similar sized fragment as expected from diagnostic PCR can be
used as a positive control for homozygous mutants. Generally, such
a control is more favorable than using an already verified mutant
strain due to differences in age or growth medium. However, we
can provide a set of mutant strains that could be useful for setting
up the procedure.

MATERIALS
REAGENTS
. Standard C. albicans strain for transformation, for example, BWP17 (relevant
genotype is arg4/arg4; his1/his1; ura3/ura3) or SN148 (arg4/arg4; his1/his1;
leu2/leu2; ura3/ura3)6,23
. pFA vectors as DNA templates for the PCR (see Figs. 1 and 2 and
Supplementary Table 1). Vector DNA is amplified in E. coli DH5a
(Invitrogen, cat. no. 11319-019) and purified using the PureYield Plasmid
Midiprep System (Promega, cat. no. A2495)
. Custom-designed oligonucleotide primers (see Supplementary Table 2):
longmers of 120 bases for the amplification of the cassettes and the
introduction of flanking homology regions, short primer pairs for the
verification of correct integration (e.g., biomers.net). Longmers should be
ordered in a medium (0.2 mmol) scale, short primers in a small (0.02 mmol)
scale. We find that HPLC purification of the oligonucleotides is sufficient for
most purposes. However, PAGE-purification is recommended, for example,
for GFP fusions. Resuspend lyophilized primers in TE to prepare a stock
solution of 10 pM ml
1. From stock solutions, prepare a 2 pmol ml
1
working solution in ddH O. All primer solutions should be stored at
20 1C
. Wizard SV Gel and PCR 2Clean-up System (Promega, cat. no. A9282;
optional)

TABLE 1 | Selectable marker genes used for C. albicans gene-function analyses.

Marker
gene Source Selection type Comments
URA3 C. albicans Auxotrophic marker; selection on This was the first selection marker available. However, owing to problems
media lacking uridine of URA3 expression and potential genome rearrangements during
counter-selection, the use of this marker has limitations18,24
Counter selection with 5¢-fluoro-orotic
acid

ARG4 C. albicans; Auxotrophic marker; selection on Preferably used as heterologous marker in C. albicans. Useful for generating
C. dubliniensis media lacking arginine deletion mutants to be tested in virulence assays23

HIS1 C. albicans; Auxotrophic marker; selection on Preferably used as heterologous marker in C. albicans. Useful for generating
C. dubliniensi media lacking histidine deletion mutants to be tested in virulence assays23

LEU2 C. dubliniensi Auxotrophic marker; selection on Heterologous selectable marker gene also useful in virulence assays23
media lacking leucine

SAT1 Synthetic Antibiotic resistance; selection on media Dominant selectable marker. Useful for wild-type isolates in conjunction
containing nourseotricine (clonat) with the SAT1-flipper22

NATURE PROTOCOLS | VOL.3 NO.9 | 2008 | 1417

 PROTOCOL
. Taq-DNA polymerase, 10 Taq-buffer (including 15 mM MgCl2), 25 mM
MgCl2 supplied with the Taq-polymerase (Genaxxon, cat. no. M3001.5000)
. dNTP oligonucleotide mix (Genaxxon, cat. no. M3313.2500)
. Primer resupension buffer (TE): 10 mM Tris-HCl, pH 7.5;
0.1 mM EDTA)
EQUIPMENT
. SpeedCycler 96/36 (Analytik Jena, cat. no. 2296)
. Transparent Microtiter plates (Analytik Jena, cat. no. 844-70002-0)
. Transparent sealing foil for microtiter plates (Analytik Jena, cat. no.
844-70027-0; or Bio-Rad, cat. no. MSA-5001)

PROCEDURE
Amplification of gene-targeting cassette
1| Set up 10 ml of Rapid-PCR mixture as outlined below. To obtain sufficient amounts of the pFA cassettes for a standard
C. albicans transformation, it will be necessary to pool six 10 ml reaction mixtures. If suitable equipment is not available for
Rapid-PCR, use standard PCR as described in Box 1. It is recommended to generate master mixes in microfuge tubes and aliquot
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

these. Reagents should be kept on a cooler.

Reagent Amount (ll) Final concentration

pFA-plasmid template (10 ng ml
1) 1 1 ng ml
1
10 Taq polymerase buffer (including 15 mM MgCl2) 1 1
MgCl2 (25 mM) 1 Total of 4 mM MgCl2
dNTPs (2 mM stock) 1 0.2 mM
S1-primer (2 pmol ml
1) 1 0.2 mM
S2-primer (2 pmol ml
1) 1 0.2 mM
Taq-DNA polymerase (5 U ml
1) 0.1 0.5 U
ddH2O 4

m CRITICAL STEP The additional amount of MgCl2 is critical to the success of Rapid-PCR; it ensures rapid polymerization in the
small reaction volume. If omitted, PCR yields will be very low or PCR will fail.
m CRITICAL STEP Remember to include positive and negative control reactions appropriate to the experiment, using suitable
primers and templates (see INTRODUCTION). If PCR results with longmer S1 and S2 primers are not satisfactory, control
amplifications can be carried out with standard primers, which are short primers that are complementary to the annealing region of
the cassettes and amplify a fragment of similar size.
2| Before transferring the PCR mixture to microtiter plates, run a plate adaptation program with this microtiter plate. This is
done by heating the microtiter plate to 99 1C for 3 min in the PCR machine. This is necessary to adapt the microtiter plate to
the heat block to ensure rapid and uniform temperature exchange in the plate. For whole-cell PCR (see Step 7), this step can be
used to break open the cells.
3| Transfer the PCR mixture to the adapted microtiter plate from Step 2 and amplify using the conditions tabulated
below. The complete set of pFA modules can be readily amplified using these standard reaction conditions (Fig. 1).

Cycle Denaturation Annealing Polymerization

1 120 s at 95 1C — —
2–41 10 s at 95 1C 10 s at 52 1C 30 s (or 45 s) at 72 1C
42 — — 120 s at 72 1C; storage at 12 1C

m CRITICAL STEP For fragments up to 2,500 bp, use a 30 s polymerization step. For fragments of 2,500–4,000 bp, use a 45 s
polymerization step.
4| Pool the six identical PCR mixtures for one transformation reaction and verify the amount of PCR product by gel electro-
phoresis using 5% of the product (3 ml). No further purification or precipitation of the product is required. However, fast and
efficient PCR clean-up systems are available (e.g., Wizard SV Gel and PCR Clean-up System, Promega, cat. no. A9282) that can
be used if desired.
’ PAUSE POINT The PCR mixtures can be stored for later transformation at
20 1C for up to 6 months.
? TROUBLESHOOTING

Transformation and selection of transformants

5| Transform C. albicans with the remaining 57 ml of PCR product according to standard protocols, either using the lithium
acetate procedure or electroporation, and select on the appropriate plates13,27,34. Transformants should appear after 2–4 d of
incubation at 30 1C.

1418 | VOL.3 NO.9 | 2008 | NATURE PROTOCOLS

 PROTOCOL

Figure 2 | Transformation strategy and verification of transformants. (a) PCR

of pFA modules will yield specific transformation cassettes for the desired a Marker
genetic alteration. After transformation, the correct integration of the S1 S2
cassette into the target locus needs to be ascertained, which includes pFA cassette
verification of the 5¢- and 3¢-integration sites and, in the case of a Marker Promoter

homozygotic deletion strain, also demonstration of the absence of internal S1 S2

S1 S2prom
PCR
bands to prove deletion of the gene. (b) Correct integration of a marker gene
Marker Promoter GFP
derived from the pFA-CdHIS1 in six independent transformants was analyzed
using the longer (45-s polymerization) SpeedCycler protocol. Ethidium S1 S2GFP
pFA cassette
bromide-stained gels show the verification bands for the 5¢-flank (upper GFP Marker
panel) and 3¢-flank (lower panel).
S1GFP S2
Transformation
6| Select six transformants for each desired mutant to verify
correct integration by colony PCR. To release the cells’ DNA,
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

individually resuspend cells from each colony in 50 ml of ddH2O Target ORF b

in a microcentrifuge tube. The remainder of each colony can be M
5′ verification
1 2 3 4 5 6
used to restreak new selective plates or for inoculation of a Verification
liquid culture. Note that this method can be used with either 0.4 kb
C. albicans or, in the case of a cloning experiment, E. coli.
pFA cassette

7| Transfer 3 ml of each cell suspension into a different well G1 ×2 ×3 G4 3′ verification

M
of a microtiter plate: with this solution, you can perform both 5′ verification 3′ verification 1 2 3 4 5 6

the plate adaptation and an initial denaturation and lysis of 0.6 kb

the cells, as described in Step 2. The remaining suspension Target ORF
can be used to restreak new selective plates or for inoculation I1 I2

of a liquid culture.
m CRITICAL STEP This procedure is recommended only for PCR
products o1 kb. If the desired product is longer, cells of a colony should be resuspended in 50 ml of ddH2O in a microfuge tube,
frozen at
20 1C for 10 min and then heated to 95 1C for 10 min before use in PCR to ensure sufficient cell lysis.
8| Prepare the PCR mixes as described below for a single reaction. To confirm correct integration of the marker gene, both
flanks of the integration site need to be checked using suitable primers (see Fig. 2a).

Reagent Amount (ll) Final concentration

10 Taq polymerase buffer (including 15 mM MgCl2) 1 1
MgCl2 (25 mM) 1 Total of 4 mM MgCl2
dNTPs (2 mM stock) 1 0.2 mM
Primer 1 (2 pmol ml
1) 1 0.2 mM
Primer 2 (2 pmol ml
1) 1 0.2 mM
Taq-DNA polymerase (5 U ml
1) 0.1 0.5 U
ddH2O 2

m CRITICAL STEP Remember to include positive and negative control reactions appropriate to the experiment, using suitable
primers and templates (see INTRODUCTION).

9| Transfer the PCR mix to each well of the microtiter plate (from Step 7) and amplify using the program detailed below. Use a
45-s polymerization step; even though the expected sizes of the PCR products are below 1,000 bp and the program takes a little
longer, we have found a 45-s polymerization step to be more reliable than a 30-s step.

Cycle Denaturation Annealing Polymerization

1 120 s at 95 1C — —
2–41 10 s at 95 1C 10 s at 52 1C 45 s at 72 1C
42 — — 120 s at 72 1C; storage at 12 1C

10| Run the entirety of each PCR product on an agarose gel to check that they are of the expected size (Fig. 2b).
m CRITICAL STEP Diagnostic PCR is used to verify the integration of the selectable marker at the target gene locus. The frequency of
additional ectopic integrations of the marker gene is practically negligible30. However, as transformation procedures themselves can be
mutagenic, at least two independent transformants should be generated for phenotypic analyses. Proof of absence of ectopic marker
integration elsewhere in the genome can be obtained by genomic Southern blot hybridizations using the selectable marker as a probe35.
? TROUBLESHOOTING

NATURE PROTOCOLS | VOL.3 NO.9 | 2008 | 1419

 PROTOCOL

 TIMING
Step 1: 15 min
Step 2: 5 min
Step 3: up to 1 h, depending on the Rapid-PCR running conditions
Step 4: 30 min
Step 5: 2–4 d
Steps 6 and 7: up to 2 h
Step 8: 30 min
Step 9: 45 min
Step 10: 30 min

? TROUBLESHOOTING
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols

Troubleshooting advice can be found in Table 2.

TABLE 2 | Troubleshooting table.
Step Problem Possible cause Solution
4 No PCR product Bubbles in the 10-ml PCR volume Do not vortex the PCR master mix; pipette carefully

Concentration of MgCl2 too low Use the indicated amount of MgCl2; this is critical
for obtaining the product

Evaporation of sample Make sure that the plates are sealed tightly before
starting the PCR program

Template DNA concentration too high Either adjust the template DNA to a maximum final
or too low concentration of 2 ng ml
1 or linearize the template
vector, for example, by ScaI digest

10 No PCR product with Insufficient or too many cells Adjust cells of a liquid culture to OD600 ¼ 4 and use
whole cells 3 ml; we find that this amount of cells gives the
most reliable results

10 No PCR product from Ectopic integration Nonhomologous integration of the marker gene
transformants could result in negative PCR results

ANTICIPATED RESULTS
The Rapid-PCR protocol ensures high yield and specific amplification of transformation cassettes based on the pFA vector series
to be used in C. albicans transformation. With these PCR products and a standard C. albicans transformation, protocol
turnaround time for a single gene alteration in C. albicans is less than 1 week.

Note: Supplementary information is available via the HTML version of this article.
ACKNOWLEDGMENTS We thank Hans-Peter Saluz and Grit Mrotzek for providing an
introduction into Rapid-PCR. Work in our laboratory is supported by the Deutsche
Forschungsgemeinschaft and the European Union—Penelope.
Published online at http://www.natureprotocols.com/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
1. Hasty, P., Rivera-Perez, J. & Bradley, A. The length of homology required for gene
targeting in embryonic stem cells. Mol. Cell. Biol. 11, 5586–5591 (1991).
2. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F. & Cullin, C. A
simple and efficient method for direct gene deletion in Saccharomyces cerevisiae.
Nucleic Acids Res. 21, 3329–3330 (1993).
3. Wach, A. PCR-synthesis of marker cassettes with long flanking homology regions
for gene disruptions in S. cerevisiae. Yeast 12, 259–265 (1996).
4. Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. New heterologous modules
for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10,
1793–1808 (1994).
5. Bahler, J. et al. Heterologous modules for efficient and versatile PCR-based gene
targeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998).
6. Wilson, R.B., Davis, D. & Mitchell, A.P. Rapid hypothesis testing with Candida
albicans through gene disruption with short homology regions. J. Bacteriol. 181,
1868–1874 (1999).
7. Taroncher-Oldenburg, G. & Stephanopoulos, G. Targeted, PCR-based gene
disruption in cyanobacteria: inactivation of the polyhydroxyalkanoic acid
synthase genes in Synechocystis sp. PCC6803. Appl. Microbiol. Biotechnol. 54,
677–680 (2000).
8. Wendland, J., Ayad-Durieux, Y., Knechtle, P., Rebischung, C. & Philippsen, P.
PCR-based gene targeting in the filamentous fungus Ashbya gossypii. Gene 242,
381–391 (2000).
9. Wendland, J. PCR-based methods facilitate targeted gene manipulations and
cloning procedures. Curr. Genet. 44, 115–123 (2003).
10. Nielsen, M.L., Albertsen, L., Lettier, G., Nielsen, J.B. & Mortensen, U.H. Efficient
PCR-based gene targeting with a recyclable marker for Aspergillus nidulans. Fungal
Genet. Biol. 43, 54–64 (2006).
11. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae
genome by gene deletion and parallel analysis. Science 285, 901–906
(1999).
12. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome.
Nature 418, 387–391 (2002).
13. Gietz, R.D. & Schiestl, R.H. High-efficiency yeast transformation using the
LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).

1420 | VOL.3 NO.9 | 2008 | NATURE PROTOCOLS


14. Fairhead, C., Llorente, B., Denis, F., Soler, M. & Dujon, B. New vectors for
combinatorial deletions in yeast chromosomes and for gap-repair cloning using
‘split-marker’ recombination. Yeast 12, 1439–1457 (1996).
15. Ninomiya, Y., Suzuki, K., Ishii, C. & Inoue, H. Highly efficient gene replacements
in Neurospora strains deficient for nonhomologous end-joining. Proc. Natl. Acad.
Sci. USA 101, 12248–12253 (2004).
16. Poggeler, S. & Kuck, U. Highly efficient generation of signal transduction
knockout mutants using a fungal strain deficient in the mammalian ku70
ortholog. Gene 378, 1–10 (2006).
17. Fu, J., Hettler, E. & Wickes, B.L. Split marker transformation increases
homologous integration frequency in Cryptococcus neoformans. Fungal Genet.
Biol. 43, 200–212 (2006).
18. Fonzi, W.A. & Irwin, M.Y. Isogenic strain construction and gene mapping in
Candida albicans. Genetics 134, 717–728 (1993).
19. Brand, A., MacCallum, D.M., Brown, A.J., Gow, N.A. & Odds, F.C. Ectopic
expression of URA3 can influence the virulence phenotypes and proteome of
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols
Candida albicans but can be overcome by targeted reintegration of URA3 at the
RPS10 locus. Eukaryot. Cell 3, 900–909 (2004).
20. Sharkey, L.L., Liao, W.L., Ghosh, A.K. & Fonzi, W.A. Flanking direct repeats of hisG
alter URA3 marker expression at the HWP1 locus of Candida albicans. Microbiology
151, 1061–1071 (2005).
21. Wellington, M., Kabir, M.A. & Rustchenko, E. 5-fluoro-orotic acid induces
chromosome alterations in genetically manipulated strains of Candida albicans.
Mycologia 98, 393–398 (2006).
22. Reuss, O., Vik, A., Kolter, R. & Morschhauser, J. The SAT1 flipper, an optimized tool
for gene disruption in Candida albicans. Gene 341, 119–127 (2004).
23. Noble, S.M. & Johnson, A.D. Strains and strategies for large-scale gene deletion
studies of the diploid human fungal pathogen Candida albicans. Eukaryot. Cell 4,
298–309 (2005).
24. Gerami-Nejad, M., Berman, J. & Gale, C.A. Cassettes for PCR-mediated
construction of green, yellow, and cyan fluorescent protein fusions in Candida
albicans. Yeast 18, 859–864 (2001).
PROTOCOL
25. Gerami-Nejad, M., Hausauer, D., McClellan, M., Berman, J. & Gale, C. Cassettes for
the PCR-mediated construction of regulatable alleles in Candida albicans. Yeast
21, 429–436 (2004).
26. Gola, S., Martin, R., Walther, A., Dunkler, A. & Wendland, J. New modules for PCR-
based gene targeting in Candida albicans: rapid and efficient gene targeting using
100 bp of flanking homology region. Yeast 20, 1339–1347 (2003).
27. Walther, A. & Wendland, J. An improved transformation protocol for the human
fungal pathogen Candida albicans. Curr. Genet. 42, 339–343 (2003).
28. Schaub, Y., Dunkler, A., Walther, A. & Wendland, J. New pFA-cassettes for PCR-
based gene manipulation in Candida albicans. J. Basic Microbiol. 46, 416–429
(2006).
29. Martin, R., Walther, A. & Wendland, J. Ras1-induced hyphal development in
Candida albicans requires the formin Bni1. Eukaryot. Cell 4, 1712–1724
(2005).
30. Dünkler, A. & Wendland, J. Candida albicans Rho-type GTPase encoding genes
required for polarized cell growth and cell separation. Eukaryot. Cell 6, 844–854
(2007).
31. Martin, R., Hellwig, D., Schaub, Y., Bauer, J., Walther, A. & Wendland, J.
Functional analysis of Candida albicans genes whose Saccharomyces cerevisiae
homologs are involved in endocytosis. Yeast 24, 511–522 (2007).
32. Saiki, R.K. et al. Enzymatic amplification of beta-globin genomic sequences
and restriction site analysis for diagnosis of sickle cell anemia. Science 230,
1350–1354 (1985).
33. Roemer, T. et al. Large-scale essential gene identification in Candida albicans
and applications to antifungal drug discovery. Mol. Microbiol. 50, 167–181
(2003).
34. Kohler, G.A., White, T.C. & Agabian, N. Overexpression of a cloned IMP
dehydrogenase gene of Candida albicans confers resistance to the specific
inhibitor mycophenolic acid. J. Bacteriol. 179, 2331–2338 (1997).
35. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
1989).
NATURE PROTOCOLS | VOL.3 NO.9 | 2008 | 1421