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43 Southern blotting ? It is first proposed by Dr. Edwin Southern in Edinburgh University in 1975, and term Southern blotting is named for him. ? Major steps: electrophoresis transfer blotting molecular hybridization Southern blot analysis gel membrane Electrophoresis blotting Hybridization DNA hybridization

45 DNA 样品 DNA 探针 变性 X-ray 片 限制性内切 酶消化 琼脂糖凝胶电泳 转移印迹 胶膜两部分工作 标记 杂交 暴光 Another illustration DNA hybridization

47 Northern blot hybridization ? Can be used to identify a particular mRNAs ? The protocol is fairly similar to that describe for southern blotting except that mRNA are not needed to be digested with any enzymes ? An experimenter might carry out northern blot hybridization to ascertain the amount of a particular mRNA present in a sample rather than its size ? Moreover, northern blot hybridization might be carried out to compare the relative levels of a particular transcript between tissues of an organism bI1 bI2 bI3 bI4 bI5 Northern analysis COB RNAs in S. cerevisiae mRNA Pre-mRNAs DNA hybridization Southern and Northern blotting DNA on blot RNA on blot 1. Genomic DNA preparation RNA preparation 2.Restriction digestion - 3.Denature with alkali - 4. Agarose gel electrophoresis ? 5. DNA blotting/transfer and fixation RNA 6. Probe labeling ? 6. Hybridization (temperature) ? 7. Signal detection (X-ray film or antibody) ? DNA hybridization Blot type Target Probe Applica........