Use of GELase™ Gel-Digesting Preparation in Constructing Chromosome-Specific YAC Libraries, EPICENTRE® Biotechnologies

EPICENTRE Forum 1 (3)

|Use of GELase™ Gel-Digesting Preparation in Constructing Chromosome-Specific YAC Libraries|

M. Campbell, R. Moyzis, L. Deaven and M.K. McCormick*, Center for Human Genome Studies & Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545, and *Molecular Neurogenetics Unit, Massachusetts General Hospital, and Dept. of Genetics, Harvard Medical School, Boston, MA 02114

The technologies of flow cytometry and yeast artificial chromosome (YAC) cloning have been combined to produce chromosome-specific DNA libraries with inserts of approximately 200 kb. However, manipulating DNA of this size without damaging it is difficult. Embedding chromosomes in agarose prior to deproteinization and digestion with restriction enzymes reduces shearing or degradation of high molecular weight DNA that may result when performing these procedures with DNA in solution.

To generate sufficient numbers of YACs with the small amount of DNA (0.5-1 µg) available from chromosomes sorted by flow cytometry, ligation of insert DNA to YAC arms must be done as efficiently as possible. This is more easily accomplished if the agarose-embedded DNA is converted to an aqueous solution prior to ligation. Following ligation, the ligated products must then be separated from remaining YAC arms prior to yeast transformation. In each case, recovering intact DNA of this size from agarose must be done gently to minimize degradation and maintain biological activity.

The following procedure uses GELase™ Gel-Digesting Preparation to optimize YAC production by maximizing recoveries of intact DNA. A technique is also described that allows isolation of ligated YAC DNA from pulsed-field electrophoresis gels without the need for ethidium bromide staining (ethidium bromide-stained YACs are not functional in yeast transformation). Using this procedure, transformation efficiencies of 1.2 x 10² to 1.7 x 10³ cfu (colony forming units) per µg of sorted DNA can be expected.

Procedure

Preparation of chromosomes and digestion of DNA

1. Chromosomes were isolated from appropriate somatic cell hybrids using bivariate fluorescence-activated flow sorting.¹ Between 0.5-1.0 µg DNA (2-10 x 10⁶ chromosomes) were collected in a Beckman ultraclear centrifuge tube that had been coated with 400 µl InCert agarose (FMC Corp.). The chromosomes were recovered by centrifugation at 1,500 x g, overnight.

2. Following centrifugation, agarose remaining on the centrifuge tube wall was carefully scraped to the bottom of the tube. The tube was placed in a 68°C water bath until the agarose melted, then placed on ice until the agarose regelled. The agarose plug was removed into a 15 ml conical polypropylene centrifuge tube.

3. Agarose plugs containing chromosomes were deproteinized overnight at 50°C in ESP (0.5 M EDTA, pH 8.0, 1% sodium lauryl sarkosyl, 2 mg/ml Proteinase K). Samples may be stored at 4°C until ready for use (up to 6 months).

4. The plugs were washed in ET buffer (10 mM Tris-HCl, pH 8.0, 50 mM EDTA) containing 0.01% phenylmethylsulfonyl fluoride for 1 hr, followed by 5-6 washes in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

5. Complete digestions with Cla I or Sac II restriction enzymes were performed overnight at 37°C by adding 200 µl of the appropriate 1X restriction buffer and enzyme to the washed plug.

6. The plug containing digested DNA was washed in 3 changes of 50 mM NaCl and melted at 68°C. After cooling to 45°C, GELase Gel-Digesting Preparation (1 U/µl concentration) was added (1 µl per 100 µl plug DNA) and the mixture incubated at 45°C for 1 hr. The digested mixture was then heated to 65°C to inactivate the GELase enzyme.

Preparation and isolation of YACs

7. DNA ligations were performed by mixing 1.0 µg of phosphatase-treated, YAC vector arms pJS97 and pJS98 in equal molar ratios² into the GELase-digested plug mixture and cooling it to 37°C. T4 DNA Ligase 10X Buffer (50 µl; supplied with the enzyme) was added, followed by T4 DNA Ligase (5 µl, 150 Weiss units), and the ligation reaction mixture was allowed to cool to room temp. The mixture was incubated overnight at 16°C.

8. Ligated DNA products were size-selected using pulsed-field electrophoresis on 0.8% LMP (low melting point)-agarose using a 4 sec pulse at 150 V for 20-24 hr.

9. As explained in Figure 1, the focused DNA ligation products were located on the gel without ethidium bromide staining, then excised from the gel and washed in 3 changes of 50 mM NaCl. Each agarose slice containing a desired DNA ligation product was digested with GELase enzyme as above (step 6).

Figure 1. Size-selection of DNA ligation products by pulsed-field electrophoresis & location without ethidium bromide staining. Ligated DNA was size-selected on a 0.8% pulsed-field gel in 0.25X TBE buffer at 150 volts for 24 hr with a 4 sec pulse time. Marker lanes were cut away from the preparative lane and the separated marker lanes were stained with ethidium bromide and notched at the stained markers (focused area). The gel was then reassembled, and the location of the focused ligation products was inferred from the stained markers. The gel area in the preparative lane corresponding to the ligation products was excised, and the remainder of the gel was stained with ethidium bromide and photographed. Lane 1, markers (200 ng of a Hind III-digest of bacteriophage-lambda). Lane 2, 1/10th vol. of ligation products (marker). Lanes 3 & 5, bacteriophage-lambda concatamers (marker). Lane 4, ligation products (preparative lane).

Transformation of yeast cells

10. Following digestion with GELase enzyme, the YAC DNA sample was diluted with an equal volume of 2X STC buffer (2 M sorbitol, 20 mM Tris-HCl, pH 7.5, 20 mM CaCl₂). A 250 µl aliquot of the diluted DNA was transformed into 500 µl of YPH250 spheroplasts₃ with 25 µg sheared E. coli DNA as a carrier. Spheroplasts of YPH250 were prepared as described^4,5 and transformed as described.^6,7 The transformation efficiency was 2-3 x 10⁶ cfu per µg of pJS97 plasmid DNA.

11. Each transformant was patched on minimal medium for screening and transferred to a 96-well microtiter plate.

This work was supported by the U.S. Dept. of Energy under contract W-7405-ENG-36.

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