Pcr-based Gene Targeting In Candida Albicans

PROTOCOL

PCR-based gene targeting in Candida albicans Andrea Walther & Ju¨rgen Wendland Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark. Correspondence should be addressed to J.W. ([email protected]).

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

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

by either using mutants in which the nonhomologous endjoining 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 homology 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 recombination 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 counterselection may induce chromosomal alterations, producing additional mutations21.

PROTOCOL

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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 recombinase, 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 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 targeting 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 transformation 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 transformants 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

transformation cassettes as outlined in this protocol to the verification 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 amplification. 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 construction. We also experimented with deviating regions, which produced comparable results. For the amplification of marker–promoter modules, 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).

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

Reagent

Amount (ll)

Final concentration

pFA-plasmid template (10 ng ml
1) 10 Taq polymerase buffer (including 15 mM MgCl2) dNTPs (2 mM stock) S1-primer (2 pmol ml
1) S2-primer (2 pmol ml
1) Taq-DNA polymerase (5 U ml
1)

5 5 5 5 5 0.5

1 ng ml
1 1 0.2 mM 0.2 mM 0.2 mM 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 1 2–41 42

Denaturation 300 s at 94 1C 60 s at 94 1C —

Annealing — 60 s at 52 1C —

Polymerization — 120–180 s at 72 1C 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 1 2–11 12–41 42

Denaturation 300 s at 94 1C 60 s at 94 1C 60 s at 94 1C —

Annealing — 60 s at 45 1C 60 s at 55 1C —

Polymerization — 120–300 s at 72 1C 120–300 s at 72 1C 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 consistently (Supplementary Table 2). 1416 | VOL.3 NO.9 | 2008 | NATURE PROTOCOLS

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,

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PROTOCOL Figure 1 | Amplification of pFA cassettes using the SpeedCycler. The repertoire of pFA modules allows the amplification of cassettes with a minimal primer set. These cassettes 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% (wt/vol) agarose gel and imaged using a Pharmacia Biotach ImageMaster gel documentation unit. Size standard M is l-DNA-cleaved with PstI.

Marker

Marker

CdHIS1 CmLEU2 CaURA3 CaSAT1 CaARG4 1,4

1,6

1,6

2,2

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.

2,2

M

Promoter

CaHIS1- CaARG4- CaARG4AgTEF1p CaMAL2p CaMET3p

2,0

2,7

3,6

Marker p M

GFP

GFP Marker

CdHIS1- CdHIS1GFPGFPAgTEF1p- CaMAL2pCmLEU2 CaARG4 GFP GFP

2,5

2,9

2,6

3,2

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 URA3

Source C. albicans

Selection type Auxotrophic marker; selection on media lacking uridine

Comments This was the first selection marker available. However, owing to problems 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; C. dubliniensis

Auxotrophic marker; selection on media lacking arginine

Preferably used as heterologous marker in C. albicans. Useful for generating deletion mutants to be tested in virulence assays23

HIS1

C. albicans; C. dubliniensi

Auxotrophic marker; selection on media lacking histidine

Preferably used as heterologous marker in C. albicans. Useful for generating deletion mutants to be tested in virulence assays23

LEU2

C. dubliniensi

Auxotrophic marker; selection on media lacking leucine

Heterologous selectable marker gene also useful in virulence assays23

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

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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 these. Reagents should be kept on a cooler. Reagent

Amount (ll)

Final concentration

1 1 1 1 1 1 0.1 4

1 ng ml
1 1 Total of 4 mM MgCl2 0.2 mM 0.2 mM 0.2 mM 0.5 U

ml
1)

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

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 2–41 42

120 s at 95 1C 10 s at 95 1C —

— 10 s at 52 1C —

— 30 s (or 45 s) at 72 1C 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 electrophoresis 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

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Figure 2 | Transformation strategy and verification of transformants. (a) PCR of pFA modules will yield specific transformation cassettes for the desired genetic alteration. After transformation, the correct integration of the cassette into the target locus needs to be ascertained, which includes verification of the 5¢- and 3¢-integration sites and, in the case of a homozygotic deletion strain, also demonstration of the absence of internal bands to prove deletion of the gene. (b) Correct integration of a marker gene derived from the pFA-CdHIS1 in six independent transformants was analyzed using the longer (45-s polymerization) SpeedCycler protocol. Ethidium bromide-stained gels show the verification bands for the 5¢-flank (upper panel) and 3¢-flank (lower panel).

a

Marker

S1

S2

pFA cassette S1

S2 PCR

Marker

Promoter

Marker

Promoter

S1

S2prom GFP

S2GFP

S1 pFA cassette GFP

Marker

S1GFP

S2

Transformation

6| Select six transformants for each desired mutant to verify correct integration by colony PCR. To release the cells’ DNA, individually resuspend cells from each colony in 50 ml of ddH2O in a microcentrifuge tube. The remainder of each colony can be used to restreak new selective plates or for inoculation of a liquid culture. Note that this method can be used with either C. albicans or, in the case of a cloning experiment, E. coli.

Target ORF

b M

1

5′ verification 2 3 4 5

6

Verification 0.4 kb pFA cassette

G1 ×2 ×3 G4 7| Transfer 3 ml of each cell suspension into a different well 3′ verification M 1 2 3 4 5 6 5′ verification 3′ verification of a microtiter plate: with this solution, you can perform both 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 I1 I2 can be used to restreak new selective plates or for inoculation 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 10 Taq polymerase buffer (including 15 mM MgCl2) MgCl2 (25 mM) dNTPs (2 mM stock) Primer 1 (2 pmol ml
1) Primer 2 (2 pmol ml
1) Taq-DNA polymerase (5 U ml
1) ddH2O

Amount (ll)

Final concentration

1 1 1 1 1 0.1 2

1 Total of 4 mM MgCl2 0.2 mM 0.2 mM 0.2 mM 0.5 U

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

1 2–41 42

120 s at 95 1C 10 s at 95 1C —

— 10 s at 52 1C —

Polymerization — 45 s at 72 1C 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



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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 Troubleshooting advice can be found in Table 2. TABLE 2 | Troubleshooting table. Step 4

Problem No PCR product

Possible cause Bubbles in the 10-ml PCR volume

Solution 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 or too low

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

10

No PCR product with whole cells

Insufficient or too many cells

Adjust cells of a liquid culture to OD600 ¼ 4 and use 3 ml; we find that this amount of cells gives the most reliable results

10

No PCR product from transformants

Ectopic integration

Nonhomologous integration of the marker gene 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).

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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).

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

PROTOCOL 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 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).

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 PCRbased 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 PCRbased 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. Du¨nkler, 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).

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