Methods and reagents: Hybridization doughnuts and uninvited phages Hybridization doughnuts *********************** Randy Hardy (hardy@mighty.fccc.edu) recently wrote about his experience with `hybridization doughnuts', an apparently widespread phenomenon on Southern blots. A clear zone is sometimes observed in the middle of an intense autoradiographic band after exposure to X-ray film of blots treated with 32-P labelled probe. When performing a genomic DNA screen, this can give the misleading impression that there are two closely migrating DNA bands. At other times, a distinct doughnut-shaped hybridization blob occurs when only a single DNA band is known to be present, as is the case with PCR products or plasmids. Several netters had different theories as to why this occurs. An unlikely source of the holes is that air bubbles are being trapped between the gel and the bottom layer of the blotting-paper sheets, or between the membrane and the gel during the capillary transfer of DNA to the nylon membrane support. One person suggested that this could be avoided by rolling a glass pipette over the gel to squeeze out any trapped air bubbles before placing the membrane on top of the gel. Fully pre-wetting the membrane and at least the first two sheets of blotting paper can help reduce trapped bubbles. It was also mentioned that finger or glove prints on the membrane might block the signal. However, these hypotheses could not explain why the holes appeared exactly over the center of the DNA bands or why doughnuts repeatedly occurred in the same lanes of the blot. Others reported observing the doughnuts, but noted that they disappeared if the blots were exposed for shorter times, indicating that they were the result of a photographic effect. It was suggested that high doses of radioactivity hitting the X-ray film might cause localized bleaching or solarization of the photographic emulsion. This would lead to a colorless spot owing to a lack of black silver-salt precipitates. One netter claimed this to be the case, since the accidental loading of 100-fold excess of positive-control plasmid DNA on a gel resulted in a bleached hybridization signal. Someone else proposed that the effect may be from posterization, a well-known darkroom technique for reversing images by exposing photographic paper to short, intense flashes of light. Another idea is that contaminants such as RNA or PCR primers mixed in with the DNA sample migrate faster than the band of linear DNA, causing the center of the band to be displaced within the gel. This may be seen as a circle or I-beam shape when a cross-sectional slice is made of the agarose gel. If the gel is thick, the bands on the top and bottom surfaces of the gel may be sheared relative to one another when pressed onto the membrane during Southern transfer. This could cause an artificial doublet or doughnut-shaped DNA blot. Overloading could produce hybridization doughnuts either by causing the DNA to migrate abnormally, or by reducing the effectiveness of UV cross-linking within the center of strong DNA bands. This could occur by simply shielding the DNA that is in close contact with the membrane. The centers of the doughnuts, which contain the highest concentration of DNA, would then crosslink poorly and the DNA could be subsequently washed away during multiple rinses with SDS. This seems to be the best explanation so far, since loading of less DNA and crosslinking for longer seem to eliminate the doughnuts. Huge plaques in a lambda library ******************************** To save time and effort, many researchers purchase genomic DNA libraries rather than construct their own in the laboratory. An interesting problem encountered by Diana Horvath (ralston@rockvax.rockefeller.edu) was the discovery of very large bacteriophage plaques that were seemingly contaminating a Nicotiana tabacum (tobacco plant) genomic lambda library purchased from a commercial supplier. She found approximately 1-5 very large plaques for every 45,000 - 50,000 normal lambda-size plaques. The abnormally large plaques appeared on the plates several hours earlier than expected, growing to 4-5 mm in diameter within nine hours. Although many factors can influence the size of library phage plaques, including the insert size, host strain and plating conditions, she suspected that these were not lambda plaques, but those of a different coliphage. Netters familiar with phage thought that the extra plaques were not of the T-even variety (e.g. T2, T4 or T6), but were most likely one of the T-odd phages (T1, T3, T5 or T7) because of the large plaque size. They felt that if the contaminant was T1, the plaques could be avoided by using a T1-resistant host strain carrying a mutation in either the tonA or tonB gene. The possibility of a T phage contaminant prompted several horror stories about how T1 phage had thoroughly contaminated microbiology laboratories in various parts of the world. Since phage T1 is quite resistant to drying, dust particles could theoretically carry dormant T1 particles. Aerosols of infected cultures can thus contaminate a whole lab, making clean phage-work extremely difficult. This problem severely hampered work with any T1-sensitive E. coli strains for weeks to months in these laboratories. Cleaning up the laboratory and equipment used in screening the library could take many hours. Experienced decontaminators wrote that an effective scrub-down could not be accomplished by simply using a disinfecting solution containing hypochlorite and, sadly, that T1 particles are almost impossible to eliminate completely. One person said that mounting a shielded UV light source facing upwards somewhere high in the room helped to reduce the number of free-floating phages in their lab. Another said that contamination problems only went away after having moved to a different building. Owing to the uncertainty as to which phage was the contaminant, and upon hearing about the possible contamination of the entire lab, Dr. Horvath chose to destroy her library by autoclaving the lot. The good news is that, after confirming that the source of the large plaques was from outside her lab, she asked for and received a refund from the company involved. ******************************************************************************* 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/dec93.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1993. Methods and reagents - Hybridization doughnuts and uninvited phages. Trends in Biochemical Sciences 18(12):484-485. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************