After studying this chapter you will be able to understand: What are the higher-order structures of DNA and RNA, and what methodologies have allowed scientists to probe these structures and the functions that derive from them? | Chapter 12 Structure of Nucleic Acids to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 1 Outline Primary Structure of Nucleic Acids ABZs of DNA Secondary Structure Denaturation and Renaturation of DNA Tertiary Structure of DNA Chromosome Structure Chemical Synthesis of Nucleic Acids Secondary and Tertiary Structure of RNA 2 Primary Structure Sequencing Nucleic Acids 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 3 DNA Replication DNA is a double-helical molecule Each strand of the helix must be copied in complementary fashion by DNA polymerase Each strand is a template for copying DNA polymerase requires template and primer Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA DNA polymerases add nucleotides in 5'-3' direction 4 Chain Termination Method Based on DNA polymerase 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 5 Chain Termination Method Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide 6 Chain Termination Method Run each reaction mixture on electrophoresis gel Short fragments go to . | Chapter 12 Structure of Nucleic Acids to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 1 Outline Primary Structure of Nucleic Acids ABZs of DNA Secondary Structure Denaturation and Renaturation of DNA Tertiary Structure of DNA Chromosome Structure Chemical Synthesis of Nucleic Acids Secondary and Tertiary Structure of RNA 2 Primary Structure Sequencing Nucleic Acids 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 3 DNA Replication DNA is a double-helical molecule Each strand of the helix must be copied in complementary fashion
