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STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00447-7
10.1016/j.xpro.2024.103282
103282
Protocol
Protocol for rapid and cost-effective extraction of genomic DNA from a wide range of wild yeast species for use in PCR-based applications
Tamm Tiina tiina.tamm.001@ut.ee
12∗
Kristjuhan Arnold arnold.kristjuhan@ut.ee
13∗∗
1 Institute of Molecular and Cell Biology, University of Tartu, 51010 Tartu, Estonia
∗ Corresponding author tiina.tamm.001@ut.ee
∗∗ Corresponding author arnold.kristjuhan@ut.ee
2 Technical contact

3 Lead contact

02 9 2024
20 9 2024
02 9 2024
5 3 103282© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Summary

Isolation of amplifiable genomic DNA is a prerequisite for the implementation of PCR-based techniques. Here we present a protocol for isolating the genomic DNA from a variety of wild yeast species. This can be completed in approximately 1 h and does not require sophisticated laboratory equipment. We describe steps for growing yeast cells, genomic data extraction, and downstream assay for amplification of specific sequences from the genomic DNA. We then detail procedures for gel electrophoresis and analysis of the results.

For complete details on the use and execution of this protocol, please refer to Kristjuhan et al.1

Graphical abstract

Highlights

• Lysis of cells using buffer containing lithium acetate and sodium dodecyl sulfate

• Precipitation of total DNA using ethanol precipitation

• Amplification of desired sequences using genomic DNA as a template

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Isolation of amplifiable genomic DNA is a prerequisite for the implementation of PCR-based techniques. Here we present a protocol for isolating the genomic DNA from a variety of wild yeast species. This can be completed in approximately 1 h and does not require sophisticated laboratory equipment. We describe steps for growing yeast cells, genomic data extraction, and downstream assay for amplification of specific sequences from the genomic DNA. We then detail procedures for gel electrophoresis and analysis of the results.

Subject areas

Genetics
Microbiology
Model Organisms
Molecular Biology
Biotechnology and bioengineering
Environmental sciences
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pmcBefore you begin

The protocol below describes a rapid and robust method for genomic DNA extraction from a variety of yeast species. It has been used for molecular identification of wild yeasts isolated from natural habitats,1 and it is also suitable for genotyping of different laboratory yeast species, including Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Ogataea polymorpha, Kluyveromyces lactis, and Komagataella pastoris.2

The first part of the protocol includes the steps for the extraction of genomic DNA. The second part of the protocol describes the steps for amplification of specific sequences from genomic DNA.

Before starting, order all reagents and kits and store at appropriate temperatures (see: key resources table). In addition, prepare all necessary buffers and media (see: materials and equipment).

Institutional permissions

All experiments in this protocol must be performed according to relevant regulatory and safety rules, and the manufacturers’ instructions for handling chemicals and reagents must be followed.

Growing of yeast cells

Timing: 0.5–1 h + 2–4 days for growth

1. Streak the wild yeast strain of interest onto the Yeast extract-Peptone-Dextrose (YPD) plate using sterile flat toothpicks. Incubate the plate at 20°C for 2–4 days. See instructions and example in Figure 1.a. Apply a small sample of cells to one edge of the agar plate and spread the cells along the edge.

b. Turn the plate, take a new toothpick, and spread the cells, making three stripes across the previous streak.

c. Repeat this process three times to cover the remaining area of the plate. Use a new toothpick each time.

Figure 1 Streaking for single colonies

(A) The initial inoculum is streaked along line 1 using a sterile toothpick. With a second toothpick, three stripes are made across streak 1. This process is repeated 3 times, each time using a new toothpick. Incubate the plate for 2–4 days until separate colonies are visible.

(B) Example of a streaked plate. The yeast Papiliotrema wisconsinensis (strain EPV606K3) cells were grown on YPD at 20°C for 4 days.

Note: Alternatively, a sterile inoculation loop or re-flammable inoculation loop can be used.

Note: Incubation temperature and time are not critical for the protocol and can be adjusted according to the optimal growth conditions of particular yeast species or strains.

Optional: Instead of streaking yeast strain of interest, existing single colonies can be spread in patches of approximately 1 cm.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Chemicals, peptides, and recombinant proteins	
	
Milli-Q H2O	N/A	N/A	
Bacto yeast extract Gibco	Thermo Fisher
Scientific	Cat# 212750	
Bacto peptone Gibco	Thermo Fisher
Scientific	Cat# 211677	
D-(+)-glucose monohydrate	Thermo Fisher
Scientific	Cat# A11090.36	
Agar, bacteriological no. 1	Neogen	Cat# NCM0236A	
Lithium acetate dihydrate (LiOAc)	Thermo Fisher
Scientific	Cat# A17921.30	
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	Cat# 436143	
Tris base	Millipore	Cat# 648310	
Ethylenediaminetetraacetic acid (EDTA) disodium salt, dihydrate	Millipore	Cat# 324503	
Hydrochloric acid (HCl)	Sigma-Aldrich	Cat# 258148	
Sodium hydroxide (NaOH)	Sigma-Aldrich	Cat# S8045	
Ethanol, absolute	Sigma-Aldrich	Cat# 51976	
UltraPure agarose	Thermo Fisher
Scientific	Cat# 16500500	
RNase A, DNase and protease-free (10 mg/mL)	Thermo Fisher
Scientific	Cat# EN0531	
TBE buffer (Tris-borate-EDTA) (10×)	Thermo Fisher
Scientific	Cat# B52	
TAE buffer (Tris-acetate-EDTA) (50×)	Thermo Fisher
Scientific	Cat# B49	
	
Critical commercial assays	
	
FIREPol master mix ready to load (12.5 mM
MgCl2)	Solis BioDyne	Cat# 04-12-00125	
100 bp DNA ladder	Thermo Fisher
Scientific	Cat# 15628019	
	
Experimental models: Organisms/strains	
	
Yeast strains	Supplied by user	N/A	
	
Oligonucleotides	
	
ITS4: 5′-TCCTCCGCTTATTGATATGC-3′	White et al.3	N/A	
ITS5: 5′- GGAAGTAAAAGTCGTAACAAGG-3′	White et al.3	N/A	
LR6: 5′-CGCCAGTTCTGCTTACC-3′	Vilgalys & Hester4 Rehner & Samuels5	N/A	
LROR: 5′-ACCCGCTGAACTTAAGC-3′	Vilgalys & Hester4 Rehner & Samuels5	N/A	
EF1-1018F: 5′-GAYTTCATCAAGAACATGAT-3’ (Y = C or T)	Stielow et al.6	N/A	
EF1-1620R: 5′-GACGTTGAADCCRACRTTGTC-3’ (D = A or G or T; R = A or G)	Stielow et al.6	N/A	
	
Other	
	
Minisart syringe filter, pore size 0.2 μm	Sartorius	Cat# S7597	
Petri dishes PS, Ø 90 × 16.2 mm, with 3 vents	nerbe plus GmbH	Cat# 09-031-0020	
MiniSpin plus microcentrifuge	Eppendorf	Cat# 5453000015	
1.5 mL Flex-Tubes	Eppendorf	Cat# 022364111	
Autoclaved toothpicks	N/A	N/A	
Mastercycler nexus X2 – PCR thermal cycler	Eppendorf	Cat# 6337000027	
PCR tube stripes with caps	Eppendorf	Cat# 0030124910	
ThermoMixer F1.5	Eppendorf	Cat# 5384000020	
ZX3 advanced vortex mixer	VELP Scientifica	Cat# F202A0176	
Mini-sub cell GT horizontal electrophoreses system and PowerPac basic power supply	Bio-Rad	Cat# 1640300	

Materials and equipment

Yeast extract-Peptone-Dextrose (YPD) plates

Reagent	Final concentration	Amount	
Bacto yeast extract	10 g/L	4 g	
Bacto peptone	20 g/L	8 g	
D-(+)-glucose	20 g/L	8 g	
Agar	20 g/L	8 g	
Milli-Q H2O	N/A	To 400 mL	
Total	N/A	400 mL	
This recipe is from Sherman.7

Note: Sterilize by autoclaving at 115°C for 15 min. From 400 mL of medium with agar, up to 20 plates can be prepared using 90 mm diameter Petri dishes. The plates can be stored at 4°C for up to 6 months.

LiOAc-SDS solution

Reagent	Stock concentration	Final concentration	Amount	
LiOAc	1 M	200 mM	10 mL	
SDS	10%	1%	5 mL	
Milli-Q H2O	N/A	N/A	To 50 mL	
Total	N/A	N/A	50 mL	

• 1 M LiOAc: dissolve 5.1 g lithium acetate dihydrate in 50 mL Milli-Q H2O, filter-sterilize using 0.2 μm filter.

• 10% SDS: dissolve 40 g SDS in 400 mL Milli-Q H2O.

Note: Store stock solutions and LiOAc-SDS solution at 19°C–25°C for up to 6 months.

TE solution

Reagent	Stock concentration	Final concentration	Amount	
Tris-HCl, pH 7.5	1 M	10 mM	100 μL	
EDTA, pH 8.0	0.5 M	1 mM	20 μL	
Milli-Q H2O	N/A	N/A	9.88 mL	
Total	N/A	N/A	10 mL	

• 1M Tris-HCl, pH 7.5: dissolve 60.57 g Tris base in 400 mL Milli-Q H2O, adjust pH with 1 M HCl.

Adjust the volume of the solution to 500 mL with Milli-Q H2O.• 0.5 M EDTA, pH 8.0: dissolve 18.61 g EDTA disodium salt, dihydrate in 80 mL of Milli-Q H2O, and adjust pH of the solution with NaOH while stirring to dissolve EDTA powder. Once fully dissolved adjust the volume of the solution to 100 mL with Milli-Q H2O.

Note: Filter sterilize using a 0.2 μm filter. Store at 19°C–25°C for up to 6 months.

Alternatives: The chemicals listed in the key resources table can be replaced with identical chemicals of the same grade from different suppliers.

CRITICAL: Sodium dodecyl sulfate causes skin irritation, eye irritation, eye damage, and is harmful if swallowed. Hydrochloric acid is corrosive to the eyes, skin, and mucous membranes and may cause respiratory irritation. Sodium hydroxide is corrosive to the skin and causes serious eye damage. Wear personal protective equipment when handling.

Step-by-step method details

Disruption of cells and extraction of genomic DNA

Timing: 1 h

This section describes the steps for isolating the genomic DNA. The use of yeast colonies from freshly streaked plates increases the efficiency of genomic DNA extraction.1. Prepare the tubes and set up the heating block:a. Label 1.5 mL tubes.

b. Transfer 0.1 mL of LiOAc-SDS buffer into the tubes.

c. Set up the heating block at 70°C.

d. Prepare 0.25 mL of 70% (v/v) ethanol per sample by diluting the 96%–100% ethanol with nuclease-free water.

Note: These preparation steps should be carried out 10 min before to allow sufficient time for the heating block to warm up.

2. Scrape a matchhead size amount of yeast cells from the YPD plate and resuspend in 0.1 mL LiOAc-SDS solution. Vortex each sample for 5–10 s.

Note: Usually, one large yeast colony is enough. In the case of slow-growing yeast species and pure cultures, several colonies can be used for analysis.

3. Incubate tubes at 70°C for 10 min.

Note: In this step, the cell walls are permeabilized. Although the indicated incubation time and temperature are optimal for the protocol, these parameters are not strictly critical. Prolonged (up to 1 h) incubation of samples is not harmful, and for the majority of the yeast species, sample incubation at 19°C–25°C is sufficient for genomic DNA extraction.

Optional: RNase A (final concentration 10 μg/mL) can be added to the LiOAc-SDS lysis solution for degradation of RNA.

4. Add 0.3 mL 96%–100% ethanol and vortex for 5 s. Incubate tubes at 19°C–25°C for 5 min.

Note: In this step, the genomic DNA is precipitated.

CRITICAL: It is essential to mix the solution by vortexing.

Pause point: After this step, the samples can be stored at −20°C for several weeks.

5. Centrifuge the tubes at 14,100 × g at 19°C–25°C for 5 min.

Note: In this step, the genomic DNA together with cellular debris is collected at the bottom of the tube.

6. Remove and discard the supernatant.

7. Add 0.25 mL 70% ethanol.

8. Centrifuge the tubes at 14,100 × g at 19°C–25°C for 1 min.

9. Remove and discard the ethanol solution with a fine tip.

Note: To ensure that the ethanol is entirely discarded, it is recommended to centrifuge the tubes for an additional 30 s at 14,100 × g at 19°C–25°C and then remove the ethanol droplets that have collected at the bottom of the tube.

10. Open the tube lid and place the tubes at 37°C for 4 min to let the residual ethanol evaporate from the pellet.

CRITICAL: Do not over-dray the pellet. When this happens, the DNA does not dissolve efficiently.

11. Resuspend the pellet in 0.1 mL of TE solution and vortex.

Note: In this step, the genomic DNA dissolves in TE solution. The solution remains cloudy because it also contains cellular debris, which does not dissolve in the TE solution. The amount of genomic DNA prepared by this protocol may vary between different samples. From Saccharomyces cerevisiae, approximately 100 nanograms of genomic DNA can be extracted from 1 × 107 cells. In addition, the pellet contains a high amount of RNA that can be removed by subsequent RNase A treatment.2

Pause point: Samples can be stored at −20°C for several days or used immediately for the next step. However, as this protocol provides crude cell lysate and does not contain any additional DNA purification steps, it is not recommended to store the samples for a prolonged period due to the gradual decay of DNA.

Amplification of specific sequences from genomic DNA using PCR

Timing: 3–4 h

This section describes the steps for PCR amplification of desired sequences from the genomic DNA as a template. Primers ITS4 and ITS5 specifically bind upstream and downstream of the 5.8S-ITS region of the ribosomal RNA (rRNA) gene. The D1/D2 region of the large subunit rRNA gene is amplified using primers LR6 and LROR. The sequence of translation elongation factor 1-alpha (TEF1) gene is amplified with EF1-1018F and EF1-1620R primers.12. Set up the PCR reaction master mix.

PCR reaction master mix

Reagent	Final concentration	Amount for 1 reaction	
5x FIREPol reaction mix ready to load (12.5 mM MgCl2)	1x	4.0 μL	
10 μM primer 1	0.5 μM	1.0 μL	
10 μM primer 2	0.5 μM	1.0 μL	
Nuclease-free water	N/A	12.0 μL	
Total	N/A	18.0 μL	

Note: In this step, the ready-to-load reaction mixture was used.

CRITICAL: If the amplified DNA fragment will be used for cloning, use a high-fidelity DNA polymerase (Phusion DNA polymerase, for example).

Optional: RNase A (final concentration 10 μg/mL) can be added to the PCR master mix to degrade RNA in the sample.

13. To one PCR tube, add 18.0 μL of master mix.

14. Centrifuge the genomic DNA solution at 14,100 × g at 19°C–25°C for 1 min.

Note: In this step, the cellular debris is pelleted, and genomic DNA stays in the supernatant.

15. Transfer 2.0 μL of the genomic DNA solution into the tube containing the PCR reaction mix.

16. Set up one control reaction using 2.0 μL of TE solution instead of genomic DNA.

17. Place the tube in a thermocycler and run the following program:

Standard PCR cycling conditions

Steps	Temperature	Time	Cycles	
Initial denaturation	95°C	10 min	1	
Denaturation	95°C	20 s	25 cycles	
Annealing	55°C	30 s	
Extension	72°C	1 min	
Final extension	72°C	10 min	1	
Hold	4°C	forever		

Note: The number of cycles given in the table (e.g. 25 cycles) is appropriate for amplification of sequences that are represented by multiple copies in the genome (e.g. rDNA genes). Increase the number of cycles (e.g. up to 30 cycles) if the sequence to be amplified is present in 1–2 copies per genome.

Pause point: After the PCR reaction, the samples can be stored at −20°C for several months.

18. Prepare a 1% agarose gel as recommended by the manufacturer of the electrophoreses system.

Note: Both tris-acetate-EDTA (TAE) and tris-borate-EDAT (TBE) running buffers are suitable for DNA agarose gel electrophoresis.

19. Load 5.0 μL of each PCR reaction into separate wells of the gel.

Note: Use a DNA ladder for sizing the amplified DNA fragments.

20. Run the gel at 100 V for ∼1 h to visualize the amplified DNA fragments (see example in Figure 2).Figure 2 Analysis of DNA amplification

Lanes 1–12: amplified DNA fragments using the genomic DNA of the indicated yeast species as a template; Lane 13: PCR reaction without template; Lane 14: 100 bp DNA ladder. Top panel: amplified 5.8S-ITS region using primers ITS4 and ITS5; Middle panel: amplified D1/D2 region using primers LR6 and LROR; Bottom panel: amplified TEF1 region using primers EF1-1018F and EF1-1620R. Inverted images of ethidium bromide-stained gels are shown.

Note: The amplified DNA fragments can be used for several downstream applications. For example, the Sanger sequencing of rDNA and TEF1 loci can be used for species identification. Also, the amplified DNA fragments can be cloned into the appropriate plasmids.

Expected outcomes

This protocol is designed to use the genomic DNA for amplification of desired sequences. We used this protocol for amplification of the 5.8S-ITS region and D1/D2 region of the large subunit ribosomal RNA gene, and the translation elongation factor 1-alpha (TEF1) gene. An example of a typical PCR amplification is shown in Figure 2.

The actual sizes of PCR products vary between different yeast species. Typically, the size of 5.8S-ITS region (amplified with primers ITS4 and ITS5) is in the range of 300–1100 bp; D1/D2 region (amplified with primers LR6 and LROR) is in the range of 950–1050 bp; and TEF1 gene (amplified with primers with EF1-1018F and EF1-1620R) is in the range of 550–750 bp.

Limitations

This protocol describes a quick, simple, and cost-efficient method of genomic DNA extraction for PCR-based applications from a wide variety of yeast species. It becomes particularly useful when simultaneous handling of a large number of samples is needed, or when access to specialized equipment or reagents is limited. It requires only a tabletop centrifuge and a heating block, and the latter is optional, as the protocol works also at 19°C–25°C in most situations. The whole procedure occurs in a single tube, no enzymes or harmful chemicals are used and all required solutions can be stored at 19°C–25°C. However, it may be inefficient for DNA preparation from some yeast species. We don’t know exactly all the factors that impact the efficiency of this protocol, however, permeabilization of the cell wall is certainly one of the critical steps that influences its reliability. We have noticed that the protocol may be inefficient in the following situations.• When slow-growing yeast strains are used. Apparently, the cell wall of slow-growing yeast strains is thicker and harder to break.

• When DNA extraction is performed using yeasts streaked on YPD plates and stored for several weeks at 4°C (so-called “old” yeasts from YPD plates). It is recommended to use freshly streaked yeast strains that have not been on plates for more than one week. However, some strains can be stored on YPD plates at 4°C up to a month without any apparent negative effect on DNA extraction.

• There are yeast species that are resistant to LiOAc-SDS cell lysis protocol. In those situations, some other DNA extraction protocol should be used. Mechanical disruption of cells by bead-beating followed by phenol-chloroform extraction of DNA8 or enzymatic digestion of cell wall9 do usually work, although these methods are considerably less convenient than the protocol described here.

Troubleshooting

As this is a very robust and quick method for genomic DNA extraction, the efficiency of single steps of the protocol is usually not monitored. The only verification step of the whole protocol is the agarose gel electrophoresis and visualization of PCR products (step 20). Ideally, it should give a single distinct band at the expected size. However, if the yeast species is not known, the exact sizes of the PCR products cannot be predicted. If the PCR reaction does not give a single DNA fragment, consider the following options for troubleshooting.

Problem 1

No amplified DNA fragment is detected.

Potential solutions

The absence of amplified DNA fragments may be caused either by problems in DNA extraction or PCR reaction.

PCR reaction issues:• To exclude general PCR reaction issues, use a positive control in PCR (steps 12–17). For example, if the PCR master mix was set up to amplify the rDNA locus (step 12), test whether it works with any other genomic DNA that was prepared by a different method.

• Use a different set of primers (in step 12). This is a particularly relevant approach if the genomic DNA was extracted from unknown yeast species and rDNA or TEF1 locus was amplified. Although these loci are highly conserved throughout evolution, small divergence of the actual sequences may exist, and using a different set of PCR primers may overcome the problem. See Stielow et al.6 and Raja et al.10 for alternative primer sequences.

• Some template sequences may be hard to amplify due to their high G/C content or the formation of secondary structures. Adding 2%–5% of DMSO into PCR reactions, or using commercially available reaction buffers designated for amplification of high G/C content templates may increase the efficiency of the reaction.

• Excess of RNA (originated from the crude genomic DNA prep) may inhibit PCR reaction. Add RNase A into the PCR master mix (step 12) and include the pre-incubation step (15 min at 60°C) before starting the PCR cycle (step 17).

DNA extraction issues:• DNA extraction is more efficient from fast-growing yeast cells. Therefore, use freshly streaked plates of yeasts and try to pick cells from the edge of the colony/patch (step 2). If the yeast colonies on plates are older than one week, re-streak the strains onto a fresh plate.

• Prolonged incubation (up to 1 h) in the LiOAC-SDS solution and higher incubation temperature (up to 90°C) may increase the DNA extraction efficiency (step 3). However, some yeast species are resistant to LiOAC-SDS cell lysis protocol, and then an alternative DNA extraction method should be used.

Problem 2

Multiple amplified DNA fragments are detected in a single reaction.

Potential solutions

• This is usually caused by impure yeast culture containing a mixture of two (or more) different species. Re-streak the strain onto a fresh plate to obtain single colonies (see “growing of yeast cells” under the section “before you begin”) and dissect different strains.

• Alternatively, this may indicate the amplification of non-specific products, which occurs if the primers bind to unrelated target sequences. Increase the annealing temperature in the PCR cycle (step 17), or add 2%–5% DMSO into the PCR master mix. Keep all PCR reagents and reaction mixtures on ice until ready to load into a thermocycler, or use a hot-start DNA polymerase.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Arnold Kristjuhan (arnold.kristjuhan@ut.ee).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Tiina Tamm (tiina.tamm.001@ut.ee).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate any unique datasets or code.

Acknowledgments

This work was supported by the Developmental Fund of the University of Tartu, the Estonian Research Council science popularization grant “Isolation and identification of Estonian yeast strains,” the Estonian Research Council grants (grant numbers PRG1741 and PRG757 ), and the European Union and Estonian Research Council via project TEM-TA3.

Author contributions

T.T. and A.K. developed the protocol and wrote the manuscript.

Declaration of interests

The authors declare no competing interests.
==== Refs
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