上游充通大兽 1.The Exploration of Genes Relies on Key Tools SHANGHAI JIAO TONG UNIVERSITY Amino acid sequence (三)影印技术 斗YA9Ys罕 GGG-GACGAG.TCC-TCC-GTT-CTC* Nucleic acid sequence codon degeneracy 》探针 由胺基酸序列推得核酸序列 Synthesizing 人工合成 ofigonucleotide >杂交 PROBE:GGGGACGAGTCCTCCGTTCT 探針標以放射性核種 雜合反應 eGGGACGAGTCCTOOGTTCT 序列互补的核酸链相互间 ATCCCCTGCTCAGGAGGCAAGAGG 自动识别、自动结合形成 DNA Target gene 核酸双链。用互补DNA或 denaturation RNA探针杂交检测特异核 Single colony 酸序列灵敏度高。 Lysed
（三）影印技术 序列互补的核酸链相互间 自动识别、自动结合形成 核酸双链。用互补DNA或 RNA探针杂交检测特异核 酸序列灵敏度高。 探针 杂交 1. The Exploration of Genes Relies on Key Tools
上游充通大兽 SHANGHAI JIAO TONG UNIVERSITY 1.The Exploration of Genes Relies on Key Tools Southern blotting 跟踪特定基因的遗传情况 DNA fragments Transfer Add DNA of DNA 32p-labeled probe revealed by blotting DNA probe Autoradiography 杂交 Agarose Nitrocellulose Autoradiogram gel sheet 分离 转膜 显影 Figure 5.3 Southern blotting.A DNA fragment containing a specific sequence can be identified by separating a mixture of fragments by electrophoresis.transferring them to nitrocellulose,and hybridizing with a 32P-labeled probe complementary to the sequence. The fragment containing the sequence is then visualized by autoradiography
1. The Exploration of Genes Relies on Key Tools Figure 5.3 Southern blotting. A DNA fragment containing a specific sequence can be identified by separating a mixture of fragments by electrophoresis. transferring them to nitrocellulose, and hybridizing with a 32P-labeled probe complementary to the sequence. The fragment containing the sequence is then visualized by autoradiography. Southern blotting 跟踪特定基因的遗传情况 分离 转膜 杂交 显影
上游充通大学 1.The Exploration of Genes Relies on Key Tools SHANGHAI JIAO TONG UNIVERSITY 各种影印技术 Blotting Compass Northern (Aine et al.,1975 RNA Northwestern (Adams et al.,1984) Protein:RNA Middle Eastern Eastern Far-Western Western Eastern- (Reinhart and Malamud, (Wreschner (Towhin ot al.: 1982:Peferoen et al.,1982; (Hummler et al.,1994) Western Thomas et al.,2009) Herzberg,1984) 79；Brunette, Protein: RNA:DNA 1981) (Bogdanov et al.,1996) Post Protein Protein Lipid:Protein Translational Modification Far-Eastern (Ishikawa Taki, 2000) Lipid:PTM Southwestern (Bowen et al.,1980) Protein:DNA Southern Electrophoretic (Southern,1975 DNA Mobility Shift Assay (Garner Revzin,1981; Fried Crothers,1981) DNA:Protein
1. The Exploration of Genes Relies on Key Tools 各种影印技术
上游充通大学 SHANGHAI JIAO TONG UNIVERSITY 1.The Exploration of Genes Relies on Key Tools (四)DNA测序 Sanger双脱氧方法 DNA to be sequenced 3-GAATTCGCTAATGG 选择性终止复制能测定DNA序列 5-CTTAA Primer DNA polymerase I Labeled dATP TTP, dCTP dGTP base Dideoxy analog of dATP 3-GAATTCGCTAATGG 5-CTTAAGCGATTA H + 3-GAATTCGCTAATGG 5-CTTAAGCGA 2'3'-Dideoxy analog New DNA strands are separated and subjected to electrophoresis
1. The Exploration of Genes Relies on Key Tools （四）DNA 测序 选择性终止复制能测定DNA序列 Sanger双脱氧方法
上游充通大兽 SHANGHAI JIAO TONG UNIVERSITY 1.The Exploration of Genes Relies on Key Tools Unknown sequence Incubation of 3 AACAGOTTCAOT 5 single-stranded DNA with unknown +5TTGT 3 Labeled primer sequenco in DNA DNA polymerase synthesis reaction +dATP,dCTP,dTTP,and dGTP mixtures containing +ddATP +ddCTP +ddTTP ddGTP dideoxyribo- nucleotides TTGTCGA TTGTC TTOTCQAAOT TTOTCG 2 Products TTOTCOAA TTOTCOAAGTC TTGTCOAAG of the TTGTCGAAGTCA reactions 12 Electrophoresis of reaction mixtures Dr.Fred Sanger 0987 Number of nucleotides in fragment 65 Frederick Sanger was awarded the Lane prize in both 1958 and 1980.He is the fourth person in the world to have been awarded two Nobel 4 Autoradiography to visualize bands and Sequence of newly Prizes and the only person to a synthesized DNA deduction of 5-3 (complementary to receive both in chemistry. sequence of newly unknown sequence) synthesized DNA strand by reading order "dideoxy"sequencing technique of bands from bottom 0 to top Primer (Sanger et al.,1977) DNA双脱氧链终止法测序 Java123.net DAddison Wesley Longman.Inc
1. The Exploration of Genes Relies on Key Tools