Labeling(标记) of dna or rna probes (why labeling?) Radioactive labeling: display and/or magnify the signals by radioactivity Non-radioactive labeling: display and/or magnify the signals by antigen labeling antibody binding- enzyme binding substrate application(signal release) End labeling: put the labels at the ends Uniform labeling: put the labels internally
27 End labeling: put the labels at the ends Uniform labeling: put the labels internally Radioactive labeling: display and/or magnify the signals by radioactivity. Non-radioactive labeling: display and/or magnify the signals by antigen labeling – antibody binding – enzyme binding - substrate application (signal release) Labeling (标记) of DNA or RNA probes (why labeling?)
End labeling 5’- end labeling using polynucleotide kinase(Nk) 3-end labeling using terminal transferase 28
28 End labeling 5’-end labeling using polynucleotide kinase (PNK) 3’-end labeling using terminal transferase
5 PNpNPNPpN∴3′ oNA or RNA Alkaline phosphatase 5- NPNPNpNPNpN。.3 A-pp-P如 Polynucleotide kinase+Hx-P)ATP 5-PNpNPNPNPNPN..... 3+ A-p-p(ADP Fig. 2. 5 -End labeling of a nucleic acid molecule
How to label one end of a dna Labeling at both ends by kinase then remove one end by restriction digestion 5’ pAATTG G G CTTAAp5 30
30 How to label one end of a DNA: Labeling at both ends by kinase, then remove one end by restriction digestion ---------------------G ---------------------CTTAAp5’ 5’pAATTC G
Uniformly labeling of DNA/RNA Nick translation labeling of DNA DNase I to introduce random nicks >DNA polI to remove dNMPs from 3 to 5 and add new dNMP including labeled nucleotide at the 3 ends Hexanucleotide primered labeling of DNA: Denature DNA> add random hexanucleotide primers and DNA pol synthesis of new strand incorporating labeled nucleotide 31
31 Uniformly labeling of DNA/RNA Nick translation labeling of DNA: DNase I to introduce random nicks →DNA polI to remove dNMPs from 3’ to 5’ and add new dNMP including labeled nucleotide at the 3’ ends. Hexanucleotide primered labeling of DNA: Denature DNA → add random hexanucleotide primers and DNA pol → synthesis of new strand incorporating labeled nucleotide