High-efficiency preparation of DNA from limiting quantities of eukaryotic cells for hybridization analysis |
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| Authors: | G T Williams |
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| Affiliation: | 1. Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China;2. State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China;3. Department of Hematology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, China;4. Hunan Provincial Key Laboratory of the TCM Agricultural Biogenomics, Changsha Medical University, Changsha 410219, China;1. Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing 400016, P.R. China;2. Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325015, P.R. China;3. Key Laboratory of Precision Diagnosis and Treatment for Hepatobiliary and Pancreatic Tumor of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, P.R. China |
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| Abstract: | This report describes a DNA preparation method which allows the detection of single-copy genes in samples of as few as 6000 eukaryotic cells. The technique uses proteinase K digestion in detergent and low-gelation-temperature agarose followed by solidification of the agarose and removal of the detergent by diffusion. RNase and restriction enzyme digestion are carried out in solution after remelting the agarose. The procedure can be performed successfully with mammalian cells in suspension, with parasitic protozoa and with pieces of mammalian tissue weighing less than 1 mg. Numerous samples can be processed simultaneously using frozen as well as fresh material. |
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