BIOTECHNOLOGY TECHNIQUES Volume 9 no.3 (March 1995) pp.225-230 Received as revised 3rd February

A SIMPLE METHOD FOR THE DIRECT EXTRACTION OF

PLASMID DNA FROM YEAST

Michael A. Sobanski” and J. Richard Dickinson School of Pure and Applied Biology, University of Wales College of Cardiff, PO Box 915, Cardiff, CFl 3TL, U.K.

Summary A rapid and simple method for the small scale isolation of shuttle plasmid

DNA from Saccharomyces cerevisiae is described. It uses glass beads to break cells and reagents which are also used in bacterial mini-preps to yield plasmid DNA without chromosomal contamination in sufficient quantities to enable direct visualisation on agarose gels.

Introduction

There are several procedures available for the purification of total DNA from

S’accharomyces cerevisicc~. These are either enzymatic treatments (,Cryer e/ ul., 1975;

Naysmyth & Reed, 1980; Sherman et al., 1986) or mechanical methods (Hoffman &

Winston, 1987; Lorincz, 1984; Ward, 1990). The scheme described here is an

improvement of the procedure of Lorincz ( 1984) which uses glass beads. This suffers

from the disadvantage that plasmid DNA is recovered together with contaminating

chromosomal DNA and hence appropriately restricted plasmid (isolated according to

size using agarose gel electrophoresis) cannot be distinguished on agarose gels. It is

therefore necessary to transform a strain of Ikhericiziu coli with various aliquots of

the extracted sample for the recovery of plasmid DNA (Ferguson et ul.. I 98 1;

Hoffman & Winston, 1987; Lorincz, 1984). Other rapid isolation procedures resulting

in clean plasmid preparations (Caldwell & Becker, 1993; Ward, 1990; Ward, 1994)

may become rather laborious and time consuming as these also rely on a subsequent

transformation step. The protocol described here removes chromosomal contaminants

(dispensing with the need for a bacterial transformation) and can be completed in less

than 65 min. This technique was found to be most useful for screening large numbers

of yeast transformants to quickly analyse the transforming plasmid DNA

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Materials and methods

Yeast strains and growth conditions The S. cerevisiae strains DBY746 (M7.a his3-1 leu2-3 leu2-I I2 trpl-289

ura 3-52) containing plasmid pMAS1 and DBY747 (MA Ta his3-I 1~2-3 leu2-I I2 trpl-289 ura3-52) containing pAYE4(34) were used throughout. Plasmid pMAS1 was constructed in our laboratory using the expression vector pYEULCB (Macreadie et al., 1991) and the PYKI (pyruvate kinase) coding region present on pAYE4(34) (Macnally et al., 1989).The YYKI gene was cloned, inframe as a 3.8 kb insert, into the Bum HI site of pYEULCB immediately downstream from the CIJPI promoter. The two different yeast shuttle plasmids used are 2 urn-based plasmids and so are maintained at medium-to high-copy number within yeast cells. Yeast strains were grown for 2-3 days at 30°C in separate patches measuring approximately 20 x 30 mm on synthetic minimal agar (0.5% ammonium sulphate, 0.16% Difco yeast nitrogen base without amino acids and ammonium sulphate, 2% glucose, w/v). Selective growth was on synthetic complete medium containing the necessary amino acid supplements but ommiting either leucine or uracil to ensure maintenance of plasmids (plasmid pMAS1 carries the S. cerevaiae iJRA3 gene and pAYE4(34) contains the S. cerevisrae lX~J2 gene which were used as the selectable markers in yeast). DNA extraction

Two-three Ioopfuls of yeast cells (taken direct from patches on minimal agar) were aseptically suspended in 200 1.11 of solution I (100 mM NaCI, 10 mM Tris-HCI pH 8.0, 1 mM EDTA, 0.1% SDS, w/v) in a 1.5 ml microcentrifuge tube. Acid-washed glass beads (0.4 mm diameter, sterilized at 160°C overnight) were added until just below the level of the liquid and vortexed at maximum speed for 2 min. Ice cold solution II (0.2 M NaOH, 1% Triton X-100, w/v) (200 ul) was then added. After mixing by inversion, 150 ul of ice cold solution III (3 M sodium acetate, pH 4.8) was added and the sample was again mixed thoroughly. Following a 5 min. incubation on ice the sample (still containing the glass beads) was treated with an equal volume of phenol chloroform-isoamyl alcohol (25:24: I, vivj. This mixture was briefly vortexed and centrifuged for 2 min. in a microfuge. The aqueous upper phase was transferred to a second tube and the phenol chloroform extraction was repeated. The aqueous layer was then placed in another microcentrifuge tube and the nucleic acids precipitated with 2 volumes of ethanol (-20°C) plus 0.1 volume of 3 M sodium acetate, pH 6.0. After 30 min. incubation on ice (or longer if desired) the DNA was pelleted by centrifugation at room temperature for 10 min. This pellet was washed with 1 ml 70% ethanol (v/v) and carefully dried. Plasmid DNA was resuspended in 80 ul TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) and stored at 4°C.

All three solutions can be sterilized by autoclaving and can be stored for several months without deterioration. Solutions II and III were stored at 4°C and solution I at room temperature. Restriction digests and anarose gel electroohoresis

Twenty microlitres of the DNA sample were digested with 12 units of the restriction endonuclease and 2~1 RNAase (10 mgiml) for 2 h at 37°C. The DNA fragments were separated electrophoretically on a 1% agarose gel made in TBE (89 mM Tris-HCI, 89 mM boric acid, 2.5 mM EDTA, pH 8.3).

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Results and Discussion

The results obtained using this procedure are shown in Fig. 1. As indicated the

plasmid DNA extracted from both strains can be successfuly cut with restriction

enzymes and isolated on agarose gels with minimal chromosomal contamination.

After the mechanical breakage of cells with glass beads, the addition of solution II

denatures double stranded DNA (caused by the strong alkaline conditions which

disrupt base pairing in the double stranded molecule). The use of solution III,

neutralises the preparation and causes rapid reassociation of DNA strands. Denatured

closed circular plasmid DNA remains interwoven and so will reanneal effectively

when conditions return to normal. Chromosomal DNA, however, will only partially

reassociate and loose chromosomal-protein aggregates form which are removed in a

subsequent centrifugation step.This method therefore has similarities with techniques

used in minipreparations of plasmid DNA from bacteria (Sambrook Ed al., 1989) and

hence is cost effective as the same solutions used in bacterial preparations can be

used with yeast. Moreover, plasmid DNA is recovered in sufficient quantities to allow

examination on agarose gels and therefore bypasses the need for competent cells

neccesary in a bacterial transformation to “bulk up” the plasmid prior to subsequent

analysis. With the method of Ward (1990), which is similar in principle to the one

presented here, purified plasmid DNA when used for bacterial transformations gave

higher transformation frequencies than when other methods were used. This was also

found to be the case when using DNA samples obtained using our protocol in

transformation experiments (DNA prepared as in Lorincz ( 1984), gave a 50 % lower

transformation frequency). Figure 2 shows the samples obtained (treated with

restriction enzyme) without the addition of solutions 11 and III during the preparation.

This provides a comparison between the DNA extract prepared using this technique

and the previous method (Lorincz, 1984).

Also examined was the effect of different vortexing times with glass beads on

the recovery ofplasmid DNA (Fig. 1). These examples show no obvious differences

in the amount of plasmid or chromosomal DNA obtained with using extended

vortexing times in the preparation of DNA extracts. 1,ongcr vortexing times may shear

genomic or high molecular weight DNA (1 loffman & Winston, 1987) howcvcr this

should not cause problems when isolating plasmids. RNA contamination was

227

negligible due to the addition of RNAase during endonuclease digestion although it

would be possible to add RNAase at an earlier stage after suspension of cells in

solution I. In summary, the method described here has advantages over other

procedures in that it can be used to identify yeast shuttle plasmid DNA direct from the

yeast (using inexpensive and convenient reagents) and hence is suited to the rapid

screening of transformed cells.

FIG. 1. Panel A: Agarose gel electrophoresis of pMAS1 plasmid DNA extracted from strain DBY746. Lanes: 1 and 4, Lambda DNA Hind III standard; 2 and 3, pMAS1 Ram HI restriction digest. Plasmid DNA was prepared after various vortexing times with acid washed glass beads (lanes 2-3: 2 min. and 3 min. respectively). Panel B : Agarose gel electrophoresis of pAYE4(34) plasmid DNA extracted from strain DBY747. Lanes: 1, Lambda DNA Hind III standard; 2 and 3, pAYE4(34) Ham HI restriction digest showing linearized plasmid. Again, lanes 2 and 3 demonstrate the effect of extended vortexing times on plasmid recovery (2 min. and 3 min. respectively).Alongside each photograph is a schematic representation of the important details.

228

FIG. 2. Agarose gel electrophoresis of Barn Hl restricted DNA extracted using the new protocol but without the use of solutions II and III during the procedure (both samples were subjected to vortexing for 2 min.). Lanes: 1, Lambda DNA Hind III standard; 2, Restriction digest of DNA extracted from DBY746 containing pMAS 1; 3, Restriction digest of DNA obtained from DBY 747 containg pAYE4(34). Alongside the photograph is a schematic representation of the important details.

Acknowledgements

We thank Dr. Ian Macreadie (CSIRO, Parkville, Victoria, Australia) for

supplying plasmid pYEXILCB and G.D. Searle and Co. (Skokie, Illinois, U.S.A.) for

plasmid pAYE4(34). M.A.S. was supported by a studentship from the Biotechnology

Directorate of the Biotechnology and Biological Sciences Research Council.

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