Lehninger Principles of Biochemistry - Chapter 8, Nucleic Acids PDF

David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 8 Nucleotides and Nucleic Acids © 2008 W. H. Freeman and Company Nucleotides Nucleotide bases Nucleotides & Nucleosides The Ribose sugar Mod...

David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 8 Nucleotides and Nucleic Acids © 2008 W. H. Freeman and Company Nucleotides Nucleotide bases Nucleotides & Nucleosides The Ribose sugar Modified bases in nucleic acids Phosphodiester bond Why DNA is more stable? Hydrogen bonding Base stacking Experiments that Proved DNA is Our Genetic Material 1. Frederick Griffith’s experiment with bacteria (Streptococcus pneumoniae) (1928) Experiments that Proved DNA is Our Genetic Material 2. Avery–MacLeod–McCarty experiment (1944) 3. Hershey & Chase experiment (1952) Chargaff’s rule  Base composition of DNA Chargaff’s rule: A always pairs with T and G pairs with C Hence the two strands become complementary to each other  Some key features of DNA In DNA, two nucleic acid strands anneal together through extensive inter-strand H-bonding between the bases. This base pairing follows the rule proposed by Watson and Crick. Chargaff’s rule: A always pairs with T and G pairs with C Hence the two strands become complementary to each other Directionality of two strands is opposite: one is 5’-3’ and another is 3’-5’ Hence complementary DNA strands are antiparallel denaturation and annealing Hyperchromic effect Hypochromic effect Laboratory methods for nucleic acid analysis DNA Gel Electrophoresis  DNA Gel Electrophoresis Agarose gel is used to analyze a mixture of DNA. DNA is negatively charged and hence migrates towards the positive terminal in the applied electric field gradient. Different DNA molecules separate according to mass. Smaller molecules migrate faster. DNA is visualized in the gel by staining with ethidium bromide, which fluoresces under UV light. UV light at 302 nm or 365 nm wavelengths are used. Polymerase Chain Reaction (PCR) and Its Applications  What is PCR? PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. It was invented in 1983 by Dr. Kary Mullis Nobel Prize in 1993 Polymerase Chain Reaction (PCR) Why “Polymerase” ? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. Why “Chain” ? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.  Polymerase Chain Reaction (PCR) The “Reaction” Components: 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction (eg. Taq polymerase) 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution - maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme  Reaction Cycle and the Machine Cycle-1 Cycle-2 (x 20-35) Cycle-3 Temperature Time 30 cycles = 230 i.e. 1,073,741,824 copies PCR tube Thermocycler