Rapid and convenient method of autoradiography for DNA cloning using digital imaging analysis
Yutaka Taketani, Mika Chikamori, Keiko Tanaka, Hironori Yamamoto,
Kyoko Morita, Ken-ichi Miyamoto, and Eiji Takeda
Department of Clinical Nutrition, The University of Tokushima School of Medicine, Japan
Abstract:Digital image analysis has been used for various biochemical and molecular biological analyses instead of autoradiography with X-ray film. However, in such cases the data manipulated by an imaging analyzer was generally printed out on normal printing paper. With normal paper, it is difficult to align the signal or its position relative to the original sample. Here in, we demonstrate it to be convenient and accurate to align signal obtained by imaging analyzer with OHP film. J. Med. Invest. 45:111-113, 1998
Keywords:screening, digital image analysis, cloning, autoradiography
Use of digital imaging analysis in Northern blotting, Southern blotting and thin layer chromatography markedly decreases exposure time and enhances sensitivity compared to conventional autoradiography. We have used the Fujix Bio-imaging-analyzer BAS2000 (Fuji Photo Film Co., Ltd., Kanagawa, Japan), instead of routine autoradiography, because of its high sensitivity (100-fold or more that of X-ray film) and resolution (100 or 200 μm). However, this system is not suited for analyzing plaque and colony hybri-dization. Since the results are printed on normal paper with a laser printer, it is difficult to ensure that orientation markers in the paper are aligned with the original plate especially on screening for cDNA or genomic DNA cloning. On the other hand, hybridization signals on X-ray film are easily aligned with the plate. To circumvent this problem, we used OHP film instead of normal laser printer paper.
MATERIALS AND METHODS
DNA cloning was performed as previously described (1-3). A human genomic DNA library in λEMBL3 (Clonetech, CA, USA) was plated on LB/agar plates at 2x104 plaques/100mm dish. Plaques were trans-ferred to Hybond C Extra nitrocellulose membranes (Amersham, UK), denatured by alkaline treatment, and fixed by baking at 80°C for 2 hours. Prehybridization was carried out at 65°C for 4 hours in hybridization buffer (50% formamide, 5X SSPE, 5X Denhardt’s, 0.1% SDS, 0.1mg/ml salmon sperm DNA). Hybridi-zation was carried out at 42°C for 12 hours in hybridi-zation buffer containing radiolabeled probe. The cDNA probe for the human sodium-phosphate cotransporter gene (NPT-1) (4) was prepared using the Megaprime DNA labeling kit (Amersham, UK) with [α-32P] dCTP (110TBq/mmol:Amersham,UK) according to the manufacture’s instructions. Washing was carried out as follows : 2X SSPE, 0.1% SDS, room temperature, 5minutes, three times;0.5X SSPE, 0.1% SDS, 55°C, 10minutes, three times. After washing off excess of radiolabeled probe, membranes were wrapped in plastic wrap and exposed to a digital analysis imaging plate (IP), instead of X-ray film, for 2hours. Following exposure, the imaging plate was set in an IP magazine and scanned with the BAS2000. Scanning parameters were as follows:gradation, 1024;resolution, 200μm;sensitivity, 10,000;latitude, 4. The scanned data were analyzed and printed out on OHP film (210x297mm, polyester). Then the same membrane was reexposed to conventional X-ray film for 3days at -80°C for comparison with the data from BAS2000.
RESULTS AND DISCUSSION
Figure1A shows the results of autoradiography with the Imaging-analyzer for the first plaque hybridi-zation screen. A positive signal was seen clearly on OHP film following 2hr exposure. In contrast, exposure for 3 days was required to detect the same signal on X-ray film at a similar intensity (Figure1B). As shown in Figure1, the signal obtained with OHP film was clearer with lower background than that seen with conventional X-ray film. These results indicate that this method markedly reduces exposure time and minimizes background compared to conventional autoradiography while keeping several of the advantages of X-ray film.
The use of X-ray film to visualize and retain a permanent record of data is essential in many research applications. However, the quality of an autoradiograph and the signal strength are often not known until the film has been developed. A high background on the film reduces the signal/noise ratio and makes data interpretation difficult. Such a situation may lead to repetition of experiments solely to obtain publication quality data. Here we describe and recommend a rapid, convenient, and accurate method for autoradiography using digital image analysis with OHP film. With this method the signal is more visible and the data interpreted more easily than with conventional X-ray film. The method is particularly applicable to initial low-stringency screening, which frequently gives rise to low intensity positive signals. The method also is applicable to Northern and Southern blotting and is extremely useful in minimizing unnecessary repetition of experiments.
We are grateful to Professor Shozo Yamamoto of the Department of Biochemistry, The University of Tokushima School of Medicine for providing access to the Bio-imaging-analyzer BAS 2000. This work was supported by Grants-in-aid from the Ministry of Education, Science and Culture of Japan.
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4. Miyamoto K, Tatsumi S, Sonoda T, Yamamoto H, Minami H, Taketani Y, Takeda E:Cloning and functional expression of a Na+-dependent phosphate co-transporter from human kidney:cDNA cloning and functional expression. Biochem J305:81-85, 1995
Received for publication July 9, 1998 ; accepted July16, 1998.
1 Address correspondence and reprint requests to Ken-ichi Miyamoto Ph.D., Department of Clinical Nutrition, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima770-8503, Japan and Fax:+81-886-33-7094.