Chapter 3 Nucleic Acids Section 2 Structure of nucleic acids
Chapter 3 Nucleic Acids Section 2 Structure of Nucleic Acids
Outline 12.1 Primary Structure of Nucleic Acids 12.2 ABZS of DNa Secondary structure 12.3 Denaturation and renaturation of dna 12.4 Tertiary Structure of DNA 12.5 Chromosome Structure 12. 7 Secondary and tertiary structure of RNA
Outline • 12.1 Primary Structure of Nucleic Acids • 12.2 ABZs of DNA Secondary Structure • 12.3 Denaturation and Renaturation of DNA • 12.4 Tertiary Structure of DNA • 12.5 Chromosome Structure • 12.7 Secondary and Tertiary Structure of RNA
Primary structure Sequencing Nucleic acids核酸序列测定 Chain termination method(dideoxy method) developed by f. sanger Base-specific chemical cleavage, developed by Maxam and gilbert Both use autoradiography -X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules
Primary Structure Sequencing Nucleic Acids 核酸序列测定 • Chain termination method (dideoxy method), developed by F. Sanger • Base-specific chemical cleavage, developed by Maxam and Gilbert • Both use autoradiography - X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules
stranded DNA DNA polymerase Primer dATP 3-OH d dGTP Annealing of primer creates a short stretch of double- stranded DNA
Chain termination method Based on dna p0小 Ferase聚合酶 reaction Run four separate reactions Each reaction mixture containS dATP, dgtp dctP and dttp. one of which is P-32-abelled Each reaction also contains a small amount of one dideoxynucleotide: either ddatp ddgtp. ddctp or ddTtp
Chain Termination Method Based on DNA polymerase 聚合酶 reaction • Run four separate reactions • Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled • Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP
branded 3' UHEULHET One dNTP is a dctP dTTP 4 reaction ekEs ::::: HA'TGCGAGACT dGCGAGACT T Gelelectrophoresis ddGATGCGAGACT ddGACACT dC GATGCGAGACI Reading sequence bMom to topi-A-G-C-G-T-A-G-C Its complement b the ortal teple Maid (-sB-T-C-G-C-A-T-C-G
) 5 OH ② PI( OH OH Primer strand A T G CC G 3′ AC G TA C T Template strand O-P-0-P-0-P-0-cH2O Base O HH ddNTP Analog b
Colored Primer dye label Template of unknown sequence DNA polymerase 4 dNTPs, one ddNTP (C) C C C C Denature C C Dye-labeled segments of DNA C spied from CoI C template with C unknown sequen
ddATP ddttP ddGtP ddcTP DNA DNA DNA DNA dNTPs dNTPs dNTPs dNTPs 5 TA TAGTA Each fragment is terminated by a dda TAGTACA TAGTACAGTA TAGTACAGTAGTTCA TAGTACAGTAGTTCAGA
5’ 3’ ||||||||||||||| ddATP DNA dNTPs ddTTP DNA dNTPs ddGTP DNA dNTPs ddCTP DNA dNTPs A T C A T G T C A T C A A G T C T A G C A C T A T A G T A T A G T A C A T A G T A C A G T A T A G T A C A G T A G T T C A T A G T A C A G T A G T T C A G A • Each fragment is terminated by a ddA
Primer 5 OH 3 CTAAGOTCGACT Template dcTP. dGTP dATP dTTP + ddATP ddCTP + ddGTP t ddTTP GATTCGAGCTGddA GATTCGAGddC GATTCGAGCTddG GATTCGAGCddT GATTCGddA GATTddc GATrCGAddG GATed GodA GATTCddG GAdd ddG C G T 11 10 987654321 AGTcGAGcTTA
