Methods and reagents: Shearing DNA for genomic library construction Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses the pros and cons of various techniques used to shear DNA for shotgun cloning. For details on how to partake in the newsgroup, see the accompanying box. Cloning of random genomic DNA fragments for library construction is usually done with DNA that has be physically broken into segments of approximately equal size. The jagged ends of the sheared DNA are then repaired enzymatically and ligated into the appropriate vector. The range of fragment sizes is typically chosen to be near the average length of a single gene within the organism of interest for gene analysis, or chosen based on the method used to automatically generate sequence data for the entire library. Maximum fragment length can also be dictated by the insert-size limitation of the chosen vector. Chop chop ********* Recently, Ian York (iayork@panix.com) asked netters for their opinions on the best methods for shearing DNA to be used for a shotgun cloning experiment. Netters suggested that the DNA could be passed through a large-gauge needle (number 18-30) several times to physically tear the DNA into fine pieces, or that the DNA could be sonicated or blended in a mini-bead beater, a small Waring blender with a teflon agitator that grinds the DNA together with microscopic glass particles added as an abrasive. It was also suggested that the DNA could be enzymatically digested with DNase I or other nucleases. One person wrote that autoclaving or boiling genomic DNA is done routinely in his lab to fragment it. However, most netters disagreed that this is a good method, as boiling the DNA would most likely only complicate matters by causing it to become single-stranded through thermal denaturation, rather than breaking it at the phosphate backbone. Because this would make the ligation of DNA fragments inefficient for cloning, netters prefer that heat treatment be reserved for DNA to be used as competitor within hybridization studies. The main problems with shearing of DNA by all of the methods mentioned, are that it can be quite difficult to control the length of the DNA fragments, and that the range of fragment sizes is generally too broad for most experiments. An elaborate protocol for efficiently obtaining the desired size of fragments by using a high-performance liquid chromatography (HPLC) pump (Waters-Pharmacia, Marlborough, MA, USA) has recently been described [1]. In that study, a device that recirculates the DNA through a 63 um-sized orifice provides random fragments with > 90% recovery. Although this would be an efficient means for producing fragments on a large scale, as is needed in automated genomic sequencing projects, the major drawback of using high-end equipment, such as a sonicator, bead-beater or HPLC pump, is that these machines are generally very expensive. Because most laboratories construct a library only once or twice a year, it is difficult to justify putting up the cost for the specialized equipment. Also, the time and effort needed to set up such a system are too great an investment for a one-off experiment. Totally nebular, dude ********************* After trying several different techniques, Ian York found that a nebulizer (a medical aid that allows asthmatics to inhale aerosols by forcing liquid through a very small hole with high pressure) gave excellent results for his intended purpose, consistently producing DNA fragments of the expected and desired size. He initially used a commercial unit called the Bio-Nebulizer, which was originally shown to be useful for disrupting cells and shearing linear DNA by S. J. Surzycki (surzycks@indiana.edu) at the University of Indiana [2]. Dr Surzycki's lab has done extensive studies of the influence of buffer viscosity (percentage of glycerol), amount of applied pressure, time exposure and of some other parameters, on the size and distribution of DNA fragments created by nebulization. The nebulization of DNA is performed by adding 0.5-5.0 ug of genomic DNA to 500 ul of TE buffer (10 mM Tris, 1mM EDTA, pH 8.0) containing 25% glycerol, collecting the fine mist into a beaker as it is passed through a small hole in the unit, and then precipitating the DNA by addition of ammonium acetate and ethanol. Medium-sized fragments averaging about 1.5 kb can be obtained by nebulizing for about 90 seconds at 12-14 psi. After shearing, the DNA is end-filled using the Klenow fragment of Escherichia coli DNA polymerase and/or phage T7 DNA polymerase, before ligating it to blunt-end vector DNA. Approximately 0.5-2.0 ug of DNA is then used to create a complete library for a genomic size of 30-100 kb. A more-detailed procedure can be obtained from the Organelle Genome Megasequencing Program, which has been using this method since 1991 at http://megasun.bch.umontreal.ca/ogmp/bank.html, or from an online protocol book at the University of Oklahoma at http://www.genome.ou.edu/protocol_book/protocol_partII.html. Unfortunately, shearing DNA using the nebulization process is covered by United States Patent US 5 610 010, and commercial use (but not research) requires explicit permission from Indiana University. The Bio-Nebulizer can be obtained by writing to Indiana University, One City Centre, Suite 200, Bloomington, IN 47404, USA, Attn: Melvin J. DeGeeter, Director, Technology Transfer (Tel. +1 812 855 7842, Fax: +1 812 855 8370). Shear pleasure ************** Anick DeMoors of Ottawa University (anick@bio01.bio.uottawa.ca) wrote that the main advantage of using a nebulizer is that, unlike sonication, which tends to cause breaks within AT-rich regions, the DNA is sheared absolutely randomly. Furthermore, the size distribution is much narrower than when using other techniques - DNA fragments nebulized to average about 1 kb in length have a distribution with the majority of the material in the range of 700-1300 base pairs. In addition, the technique takes less than 15 min to perform, and it is very easy to control the size range of the DNA by altering the gas pressure blowing through the nebulizer, the viscosity of the liquid and the duration of nebulization. One more advantage is that disposable nebulizers, such as the `Up-Mist' medication nebulizer manufactured by Hospitak or a similar unit made by IPI Medical Products Inc., Chicago, IL, USA, and used for delivering respiratory drugs, are available from any medical supply store. These cost less than US$2.00 each, and the disposable units can be re-used for similar experiments if there is no concern for contaminating the DNA [3]. Ian York suggests one modification of the procedure is to place a piece of plastic tubing over the end of the output nozzle. The opening can then be further reduced in size by covering it with masking tape and slicing the tape with a razor blade to make a tiny slit. He says that this prohibits the liquid from dispersing too rapidly, and it increases the volume of liquid recovered by 10-20%, for a total recovery of 80-90% of the applied DNA. The recovery of sample is, after all, an important consideration when making libraries from scarce DNA samples. For example, only about 1-3 ug of mitochondrial DNA is all that is needed to establish a clone library because it has a relatively small genome of 50-80 kb. The drawbacks of nebulization are that it is a little more technically demanding than needle aspiration, and if the DNA has been previously mistreated or nicked by enzymes, the DNA might be sheared inconsistently. However, netters say this is not so much a problem, and would occur regardless of the shearing method used. One person did point out that care must be taken not to inhale the aspirated DNA, especially if the DNA is from a pathogenic microorganism, virus, or any malignancy, as epithelial lung cells can be transformed directly with microdroplets of DNA solution [4]. All in all, netters say that nebulization is quick and easy to learn, and that they consistently obtain good results with it. They say that to perform efficient lost-cost shearing, all you need is a nebulizer, tygon tubing and masking tape to cover the breathing hole. References ********** [1] Oefner, P. J. et al. (1996) Nucleic Acids Res. 24, 3879-3886 [2] Okpodu, C. M. et al. (1994) BioTechniques 16, 154-159 [3] Bodenteich, A. et al. (1994) in Automated DNA Sequencing and Analysis (Adams, M. D., Fields, C. and Venter, J. C., eds), pp. 42-50, Academic Press [4] Alton, E. W. F. W. et al. (1993) Nat. Genet. 5, 135-142 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jul97.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1997. Methods and reagents: Shearing DNA for genomic library construction. Trends in Biochemical Sciences 22(7):273-274. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************