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![Henderson–Hasselbalch
Equation
Water is ionizable
H2O + H2O = H3O+ + OH-
KW (equilibrium constant)
[H+] x [OH-]
[H2O]
=
HA = H+ + A-
Ka (equilibrium constant)
[H+] x [A-]
[HA]
=
pH = pKa + log10
[A-]
[HA]](https://image.slidesharecdn.com/lecture1nucleicacidemailed-170211180536/85/Nucleic-acid-9-320.jpg)
Uploaded byAnkit Kumar
Nucleic acid
This document outlines the schedule and topics for a course on the science of living systems. The course covers topics such as nucleic acids, transcription and translation, protein structure, enzymes, photosynthesis and respiration, cell division, the immune system, and recombinant DNA technology. It includes the dates for lectures, exams, and breaks. The document also provides background information on water and its importance for life, as well as DNA structure and replication. Key concepts in molecular biology such as the central dogma are explained.
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Nucleic acid
- 1. Science of Living System SoumyaDe School of Bioscience Email: somde@iitkgp.ac.in Tel: 03222-260514 BS20001
- 2. Lecture Date Topic 120/7/16 Nucleic acids 2 27/7/16 Transcription and Translation 3 3/8/16 Protein structure 4 10/8/16 Enzymes 5 17/8/16 Photosynthesis and respiration 6 24/8/16 Cellular architecture ** 31/8/16 CLASS TEST-1 7 7/9/16 Cell division and apoptosis ** 14/9 to 21/9 MID-SEM EXAM 8 28/9/16 Host Defense/Immune system 9 5/10/16 Disease biology/vaccines/antibiotics ** 12/10/16 Autumn Break 10 19/10/16 Responses of living systems/scaling factors 11 26/11/16 Recombinant DNA Technology & its impact 12 2/11/16 Advances in Biology and Biomedical Engineering ** 9/11/16 CLASS TEST-2 ** 16/11/16 DISCUSSION AND REVISION
- 3. What are thecharacteristics of a Living System? • High degree of complexity. • Mechanisms for sensing and responding to alterations in surroundings. • Capacity for precise self-replication and self- assembly. • Systems for extracting, transforming and using energy from the environment. • Ability to evolve.
- 4. Water – simplestyet most important molecule necessary for Life! • Water is the biological solvent on Earth. • Water makes up 70% or more of the weight of most organisms. • The abundance of water is the primary reason that makes Earth a habitable planet.
- 5. Water – simplestyet the most important molecule necessary for Life! 1A = 0.1 nm = 10-9m
- 6.
- 8. Hydrogen bond, ionicand hydrophobic interactions stabilize enzyme- substrate complex
- 9. Henderson–Hasselbalch Equation Water is ionizable H2O+ H2O = H3O+ + OH- KW (equilibrium constant) [H+] x [OH-] [H2O] = HA = H+ + A- Ka (equilibrium constant) [H+] x [A-] [HA] = pH = pKa + log10 [A-] [HA]
- 11. RNA: Ribonucleic Acid DNA:Deoxyribonucleic Acid Nucleic Acid
- 12. Flow of GeneticInformation: The Central Dogma of Molecular Biology RNA ProteinDNA Transcription Translation Replication RNA ProteinDNA Polymer of nucleotides Polymer of nucleotides Polymer of amino acids
- 13. • Nucleic acidsare polymers • Monomer---nucleotides – Nitrogenous bases • Purines • Pyrimidines – Sugar • Ribose • Deoxyribose – Phosphates Nucleosides Nucleotides Nucleic Acid
- 14. (Ribose) RNA - RibonucleicAcid DNA - Deoxyribonucleic Acid RNA vs DNA Ester Bond N-β-glycosyl bond (OH) (H)
- 15. Two Types ofNitrogenous Bases
- 16.
- 17. Nucleotide = Nucleoside+ Phosphate (Nucleoside = Sugar + Base)
- 18. Nucleotide = Nucleoside+ Phosphate (Nucleoside = Sugar + Base)
- 19. Phosphodiester Linkages inthe Covalent Backbone of Nucleic Acid 3′ end3′ end
- 20.
- 21. RNA is LessStable than DNA
- 22. Discovery of theDNA Structure • Structure was discovered in 1953 by James Watson and Francis Crick • Awarded Nobel Prize in 1962
- 23.
- 24. Watson-Crick Model forthe Structure of DNA a b c
- 25.
- 26. Salient Features ofA, B, and Z Forms of DNA
- 27. Reversible Denaturation and Annealing(Renaturation) of DNA
- 28.
- 29. UV Light CausesDNA Damage by Forming Thymine Dimers Kink Photolyases are DNA repair enzymes that repair damage caused by exposure to ultraviolet light.
- 30. DNA Replication: AnOverview
- 31. Semiconservative Replication ofDNA as suggested by Watson and Crick
- 32.
- 33. The Meselson-Stahl ExperimentSupports Semiconservative Replication
- 34. Outline for Replication A.Initiation B. Priming C. Elongation D. Proofreading and Termination
- 35. Worked with E.coli. Discovered the mechanisms of DNA synthesis in 1956. Four components are required: 1. dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates) (sugar-base + 3 phosphates) 2. DNA template 3. DNA polymerase (Kornberg enzyme) 4. Mg2+ (optimizes DNA polymerase activity) Arthur Kornberg: Nobel Prize in 1959
- 36. 3 Polymerase III Leading strand 5’ 5’ 3’ 3’ SupercoiledDNA relaxed by gyrase & unwound by helicase + proteins Helicase + Initiator Proteins ATP SSB (single strand binding) Proteins RNA Primer primase 2Polymerase III Lagging strand Okazaki Fragments 1 RNA primer replaced by polymerase I & gap is sealed by ligase Replication: The Basic Concept
- 37. Laboratory methods for nucleicacid analysis
- 38.
- 39. 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.
- 40. Polymerase Chain Reaction (PCR)and Its Applications
- 41. What is PCR? PCRis 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
- 42. 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.
- 43. 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 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 Polymerase Chain Reaction (PCR)
- 44. PCR Reaction Steps Double-strandedDNA Denaturing (heat to 95oC) Strands separated Annealing (Lower temperature to 55oC) Primers bind to template DNA Extension (Increase temperature to 72oC) Taq polymerase synthesizes new DNA strands
- 45. Reaction Cycle andthe Machine ThermocyclerPCR tube Cycle-3Cycle-1 Cycle-2 (x 20-35) Time Temperature
- 46. Efficiency to Solvea Forensic Problem 1copy Cycle 1 Cycle 3 Cycle 2 8,388,608 copies (223) 20 more cycles
- 47. DNA Sequencing Sequencing isthe process by which you determine the exact order of the nucleotides in a given region of DNA Dideoxynucleotide sequencing is done through complementary chain synthesis and early termination The synthesized chains are visualized by methods using: – Radioactive labels – Nonradioactive labels Frederick Sanger Nobel Prize in 1980
- 48. Requirements for Sanger-Coulson Sequencing DNAto be sequenced must be in single strand form The region to be sequenced must be 3’ flanked by known sequence Reagents needed are: – A primer complementary to the known region to direct chain synthesis. – DNA polymerase. – 4 deoxynucleotide triphosphates (dNTPs). – 4 dideoxynucleotide triphosphates (ddNTPs).
- 49. Dideoxynucleotides (ddNTPs) The 3’hydroxyl has been changed to a hydrogen in ddNTP’s, which terminates a DNA chain because a phosphodiester bond cannot form at this 3’ location
- 50. Since the 3’–OH is changed to a –H in ddNTPs, it is unable to form a phosphodiester bond by nucleophilic attack on the phosphate, and it will cause a termination in the DNA chain Mechanism of DNA polymerization
- 51. Sequencing Visualization Methods Twoforms of labeling: – Radioactive • Primer labeled (32P or 33P) • dNTP labeled (35S) – Nonradioactive • Primer labeled • ddNTP labeled (big dye terminator)
- 53. Gel Separation The reactionmixtures are separated on a denaturing polyacrylamide gel. – Denaturing gel prevents the DNA from folding up on itself while it travels through. – Polyacrylamide gel separates the strands which differ in length by only one nucleotide. Each band corresponds to a sequence of DNA which was terminated by a particular ddNTP. This ddNTP is identified by lane in the radioactive method and by color in the fluorescent method The lowest band on the gel is the shortest. The shorter the strand, the earlier in the synthetic reaction the ddNTP was incorporated. The lowest band on the gel is at the 5’ end of our synthesized strand and is complementary to the 3’ end of our unknown fragment.
- 57.
- 58. Extra resources Book: The DoubleHelix: A Personal Account of the Discovery of the Structure of DNA - By James D Watson Videos: DNA replication https://www.youtube.com/watch?v=TNKWgcFPHqw DNA sequencing: https://www.youtube.com/watch?v=vK-HlMaitnE Polymerase Chain Reaction (PCR) http://video.mit.edu/watch/pcr-polymerase-chain-reaction-10948/ https://www.dnalc.org/view/15475-The-cycles-of-the-polymerase-chain- reaction-PCR-3D-animation.html