Isolation of Intact, High Molecular Weight DNA Fragments for the E. coli Genome Project, EPICENTRE® Biotechnologies

EPICENTRE Forum 4 (3)

|Isolation of Intact, High Molecular Weight DNA Fragments for the E. coli Genome Project|

Heather A. Kirkpatrick and Frederick R. Blattner, University of Wisconsin, Department of Genetics, Madison, WI 53706, and Heidi L. Kijenski, Qiagen, Inc., Santa Clarita, CA 91355

Many of the defining experiments of modern molecular biology were carried out in Escherichia coli. Experiments in E. coli have revealed fundamental information in many areas of study such as gene regulation, drug resistance, mutagenesis, DNA rearrangement and transfer mechanisms, and signal transduction. The sequence of the E. coli genome continues to be of interest to a wide range of disciplines.

An important step in any genome sequencing project is library construction for which isolating intact, high molecular weight DNA fragments is critical. For completing E. coli,¹ we isolated sub-fragments directly from the 4,639,221 bp chromosome. BAC, YAC, or cosmid vectors are often utilized in other genome projects. Rare-cutting I-Sce I sites were inserted into the E.coli chromosome using a mini Tn-10 transposon as described by Rode, C.K., Obereque, J.H., and Bloch, C.A.² Upon digestion with I-Sce I endonuclease, a unique genomic fragment is released and isolated by pulsed field gel electrophoresis as illustrated in Figure 1. Ten unique non-overlapping genomic fragments ranging in size from 150 to 270 kb (average size 250 kb) were isolated in this manner. We used GELase™ Agarose Gel-Digesting Preparation at several steps in our cloning procedure including recovery of the genomic fragments for shotgun cloning and eventual sequencing (Figure 2). Here, we describe isolation of a 226,878 bp genomic fragment from the E. coli K-12 M1655 chromosome using GELase Preparation.

To isolate intact E. coli chromosomal DNA containing the fragment of interest, we first prepared E. coli agarose plugs using the method developed by Smith et al.³ with modifications by Chen et al.,⁴ as well as some of our own modifications. E.coli K-12 MG1655 was grown overnight to saturation, harvested, washed twice with Pett IV buffer (10 mM Tris-HCl, pH 7.6, 1M NaCl), and resuspended in Pett IV buffer at one-tenth volume of the overnight culture. The resuspended cells were mixed 1:1 (v/v) with 0.8% InCert® Agarose (FMC BioProducts) and held at 42°C while the 20 µl agarose plugs were dispensed. After solidifying, the plugs were pooled in a 50ml tube and incubated at 37°C overnight in EC lysis buffer (6 mM Tris-HCl, pH 7.6, 1 M NaCl, 100 mM EDTA, 0.5%Brij-58, 0.2% deoxycholate, 0.5% N-lauroylsarcosine, 1 mg/ml lysozyme, 20 µg/ml RNase A). The following morning the solution was exchanged with ESP buffer (0.5 M EDTA, pH 8.0, 1% N-lauroylsarcosine, 1 mg/ml Proteinase K; Note: the ESP buffer was pre-incubated for 2 hours at 37°C prior to use) and the plugs were incubated at 50°C overnight. The next day the solution was changed to fresh ESP buffer and again the plugs were incubated at 50°C overnight. The next morning the ESP buffer was replaced with TE-PMSF buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride) and the plugs were incubated at 37°C overnight. The PMSF was then removed by 30-minute washes with TE buffer (10 mM Tris-HCl, pH7.5, 0.1 mM EDTA), followed by a final overnight wash in the same solution.

To prepare the DNA-agarose plugs for digestion with I-SceI, the plugs were aliquoted into tubes (30 plugs/2.2ml tube; 10 tubes total) and equilibrated in I-Sce I soak solution [11 ml ddH₂O, 1.25 ml 10X I-Sce I buffer, 0.625 ml I-Sce I storage buffer, 5 µg Enhancer (supplied with the enzyme)]. A 10 U/µl stock of I-Sce I (Boehringer Mannheim) was diluted 1/8 with I-Sce I storage buffer. One microliter of the diluted enzyme was mixed with 29µl of soak solution and dispensed into each tube. The solution was distributed in the tubes by very brief centrifugation in a microcentrifuge and the tubes were incubated at room temperature for 1 hour to allow the enzyme to diffuse into the plugs. MgCl₂ was added to each tube to a final concentration of 50 mM and distributed by brief centrifugation as above. The tubes were then incubated at room temperature for 2 hours. After digestion, the reaction mixture was removed from the plugs and 0.5 M EDTA was added. Again the tubes were centrifuged in a microcentrifuge to distribute the EDTA. After 15 minutes, the EDTA was removed and the tubes were placed in a 70°C water bath for 10 minutes or until the plugs were completely melted.

The plugs were then loaded onto a 1% SeaPlaque® GTG® agarose gel (FMC BioProducts) in 0.5X TBE buffer. Electrophoresis was performed on a Bio-Rad CHEF-DR® III pulsed field electrophoresis system at 6 V/cm for 18 hours with a pulse time of 30 to 35 seconds. Following electrophoresis, a small vertical slice was cut from the left and right sides of the gel, stained with ethidium bromide, and viewed under UV light. The position of the 227 kb fragment was marked with a scalpel. The gel was reassembled and the 227 kb band was excised. The remainder of the gel was then stained with ethidium bromide to confirm the excision of the 227 kb fragment.

The gel slice was incubated in 50 ml of 1X GELase Buffer overnight. Following this buffer exchange step, excess buffer was removed and the gel slice transferred to a clean 50 ml tube. EDTA was added to a final concentration of 10 mM, along with the appropriate amount of 50X GELase Buffer and the tube was incubated at 70°C until the agarose was completely melted. The melted agarose was equilibrated to 45°C in a water bath, and GELase Preparation was added to a concentration of 2 units/300 mg gel. The mixture was incubated at 45°C overnight to digest the agarose. Following agarose digestion, the DNA was recovered by ethanol precipitation and ultracentrifugation.

Recovery of the ~227 kb genomic fragment from the agarose gel was approximately 80-90%. As shown in Figure 3, the DNA was recovered without degradation. The purified DNA was then ready for shotgun cloning.

Figure 3. Recovery of the 227 kb genomic fragment. The DNA fragment was isolated as described and subjected to electrophoresis on a 1% agarose gel. Lane 1, fragment prior to isolation with GELase Preparation; Lane 2, fragment recovered using GELase Preparation prior to ethanol precipitation; Lane 3, recovered fragment following both GELase treatment and ethanol precipitation.

The purification of high molecular weight DNA from agarose gels was a critical step in the cloning procedure used for the E. coli genome sequencing project. Using GELase Preparation, we were able to recover genomic fragments with high yield and minimal degradation. We have successfully processed ten fragments using this procedure which generated approximately 2.3 mb of finished E. coli sequence.

Blattner, F.R., Plunkett III, G., Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.F., Gregor, J., Davis, N.W., Kirkpatrick, H.A., Goeden, M.A., Rose, D.J., Mau, B., and Shao, Y. (1997) "The Complete Genome Sequence of Escherichia coli K-12," Science 277, 1453.
Rode, C.K., Obereque, J.H., and Bloch, C.A. (1995) "New Tools for Integrated Genetic and Physical Analyses of the Escherichia coli Chromosome," Gene 166, 1.
Smith, C.L., Econome, J.G., Schutt, A., Kleo, S., and Cantor, C.R. (1987) "A Physical Map of the Escherichia coli K-12 Genome," Science 236, 1448.
Chen, L., Kosslak, R., and Atherly, A.G. (1994) "Mechanical Shear of High Molecular Weight DNA in Agarose Plugs," BioTechniques 16, 228.