Acid nucleic

Nội dung text: Acid nucleic

1. CONTENT ❑Nucleoside, nucleotide, oligonucleotide ❑DNA, RNA ❑Physical & chemical properties ❑Modification mechanism ❑Biosynthesis of nucleotide ❑Application of molecular biology in Food technology ❑Restriction enzymes ❑Genetic engineering ❑GMO (Genetically modified organism) ❑GMF (Genetically modified food) ❑Human genome
2. NUCLEOTIDE STRUCTURE PHOSPHATE SUGAR (5C) NITROGEN BASE (C-N ring) GROUP (PO4) Ribose (RNA) PURINES PYRIMIDINES Deoxyribose (DNA) Adenine (A) Thymine (T); Uracil (U) Guanine(G) Cytocine (C) NUCLEOTIDE
3. NUCLEOTIDE STRUCTURE - BONDS
4. POLYNUCLEOTIDE-BACKBONE ❑THE SUGAR-PHOSPHATE BACKBONE ▪ Sugar-phosphate joined by phosphodiester bonds ▪ Sugar-phosphate in poly nucleotide orientated in the same direction ▪ 3’-OH group of the sugar in one nucleotide forms an ester bond to the phosphate group on the 5’-carbon of the sugar of the next nucleotide
5. NUCLEOTIDE STRUCTURE-BASE ❖Bases ▪ Attached to the 1st Carbon of sugar
6. NUCLEOSIDE & NUCLEOTIDE • Nucleotide – nucleoside forms a phosphate ester with the C5’-OH group of ribose or deoxyribose – Named: using the name of the nucleoside followed by 5’- monophosphate: adenosine 5’-monophosphate • Oligonucleotide: short nucleotide (<100 nt) (primers for DNA replication)
7. ATP - MAJOR ENERGY SOURCE FOR CELLULAR ACTIVITY ❑ ATP: adenosine triphosphate ▪ All cells need chemical energy carried out by ATP ▪ Molecules in food store chemical energy in their bonds Starch molecule Glucose molecule
8. ATP-CHEMICAL STRUCTURE + +
9. THE ADP-ATP CYCLE ATP ATP-ase Synthetase
10. WHEN IS ATP MADE? ❑During Cellular Respiration • Includes pathways that require oxygen • Glucose is oxidized and O2 is reduced • Glucose breakdown → one molecule of glucose → 36-38 ATP molecules
11. DNA STRUCTURE – DISCOVERY X-ray diffraction photo of DNA (1952) by Rosalind E. Franklin Rosalind Elsie Franklin(1920-1958), Maurice Hugh Frederick Wilkins British biophysicist and X-ray (1916-2004), New Zealand- crystallographer, born English physicist Took X-ray diffraction photo of DNA and molecular biologist (1952)
12. DNA STRUCTURE – THE NOBEL PRIZE ❑ Crick, Watson and Wilkins won the Nobel Prize for medicine in 1962 ❑ Rosalind Franklin, Maurice Wilkins’s colleague, developed the technique to photograph a single strand of DNA → died of cancer in 1958 →could not be recognized in the Nobel Award
13. WATSON & CRICK MODEL OF DNA ❑Nucleotides bond between DNA strands ▪ H bonds ▪ Purine :: Pyrimidine pairing A :: T: 2 H bonds G ::: C: 3 H bonds ▪ Arranged in step-like pairs ▪ Determines the genetic information of DNA
14. BASE-PAIRING RULE OF NUCLEOTIDE Erwin Chargaff (1905-2002), Purine – Pyrimidine pairing Austrian American biochemist, A :: T (2 H bonds) discovered the base-pairing rule of G ::: C (3 H bonds) nucleic acids
15. DNA - FUNCTION ❑Storage of genetic information ❑Self-duplication & inheritance ❑Expression of the genetic message
16. DNA - SEQUENCE ❑Reading DNA sequence ▪ The sequence: read from 5’ to 3’ end using the letters of the bases • Ex: 5’—A—C—G—T—3’ ▪ Free 5’ end: phosphate group ▪ free 3’ end: - OH group
17. DNA - SUPERCOILS ❑Each cell contains about 2 meters of DNA → DNA “packaged” by coiling around a core of proteins (histones: rich in lysine and arginine residues) ❑The DNA-histone: nucleosome ❑In eukaryotic cells (animals, plants, fungi), DNA stored in the nucleus
18. Properties of DNA D. DNA can be renatured 1. Effect of time, conentration and complexity on renaturation E. General forms of a DNA helix 1. A form 2. B form 3. Z form
19. DENATURATION & RENATURATION OF DNA
20. Structure and Function correspondence of protein and nucleic acid Protein Nucleic Acids Fibrous protein Globular protein Helical DNA Globular RNA Structural protein ⚫ Enzymes Genetic ⚫Ribosomal RNA ⚫ Antibodies information (rRNA) maintenance ⚫ Receptors, etc ⚫Transfer RNA (tRNA) ⚫Signal recognition
21. Stability 1. Hydrogen bonding • Stability lies in the stacking interactions between base pairs 2. Stacking interaction/hydrophobic interaction between aromatic base → maximized in double-stranded DNA (base stacking & hydrophobic effect)
22. Effect of Alkali • DNA denaturation at high pH keto form enolate form keto form enolate form Base pairing is not stable anymore because of the change of tautomeric (states of the bases →DNA denaturation
23. Chemical Denaturation Formamide (HCONH2) and Formaldehyde : Northern blot Disrupting the hydrogen bonding of the bulk water solution Hydrophobic effect (aromatic bases) is reduced Denaturation of strands in double helical structure
24. Renaturation Rapid cooling Slow cooling Whole complementation of dsDNA Annealing Base paring of short regions of complementarity within or between DNA strands. (example: annealing step in PCR reaction) Hybridization Renaturation of complementary sequences between different nucleic acid molecules. (examples: Northern or Southern hybridization)
25. Spectroscopy of Nucleic Acid 1. UV absorption - Nucleic acids absorb UV light due to the aromatic bases - The wavelength of maximum absorption by both DNA and RNA is 260 nm (lmax = 260 nm) - Applications: detection, quantitation, assessment of purity (A260/A280) 2. Hypochromicity Fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide
26. DIFFERENCES BETWEEN RNA & DNA ❑RNA (ribonucleic acid) ▪ Differences between RNA and DNA - Pentose sugar: ribose, [deoxyribose in DNA] - Uracil replaces thymine - Single stranded [DNA: double stranded] - Much smaller than DNA - Three main types of RNA: ✓ribosomal (rRNA), messenger (mRNA) and transfer (tRNA)
27. TYPES OF RNA
28. TRANSFER RNA (tRNA) ❑Transfer RNA ▪ Translates the genetic code from the mRNA ▪ Brings specific amino acids to the ribosome for protein synthesis ▪ Each amino acid is recognized by one or more specific tRNAs - one end: attaches to the amino acid - the other end binds to the mRNA (complimentary sequence)
29. GENETIC CODE ORGANIZATION ▪ Single-base changes (single-nucleotide polymorphism) in the third position in a codon → produce the same amino acid ▪ The second base specifies if the amino acid is polar or apolar (hydrophobic) ▪ Changes elsewhere in the codon → produce a different amino acid, but with the same physical-chemical properties
30. Ionic R groups make the amino acid hydrophilic
31. THE CENTRAL DOGMA OF MOLECULAR BIOLOGY (BY F. CRICK) Francis Harry Compton Crick (1916-2004) ▪ Replication: DNA is copied with very high fidelity ▪ Transcription: DNA genetic code is read and transferred to messenger RNA (mRNA) ▪ Translation: genetic code is converted to a protein
32. DNA REPLICATION - OVERVIEW ❑DNA Replication - Semi-Conservative Reiji Okazaki (1930-1975) Japanese molecular biologist → Discovered Okazaki fragments with his wife in 1966 Okazaki fragments • 1,000-2,000 nu. long in E. coli • 100-200 nu. long in eukaryote • separated by ~10 nu. RNA primers
33. PROOFREADING AND REPAIRING DNA 1 A thymine dimer distorts the DNA molecule. 2 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease DNA 3 Repair synthesis by polymerase a DNA polymerase fills in the missing nucleotides. DNA ligase 4 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete.
34. DNA DAMAGE ❑Physical factors ▪ ionizing radiation: ▪ inducing a large number of free radicals →lead to DNA damage ▪ rupture of phosphodiester bond → DNA breaks ✓ Application: radiation therapy of cancer: using ionizing radiation → damage the DNA of cancer cells →stop its division → cell death
35. DNA DAMAGE ❑Chemical factors ▪deamination damage of DNA bases: cytosine, adenine and guanine bases with exocyclic-amino (-NH2) → removed under the influence of water, oxidants and free radicals and some other substances →turning the cytosine into uracil, →Error in the replication (bases-mismatched) → gene mutation ➢ Nitrite induce the deamination of adenine to form hypoxanthine → paired with cytosine in the DNA replication → resulting in A • T → G • C conversion
36. MUTATION ▪ Frame shift mutation ✓Insertions or deletions of nucleotides →Change reading frame ❖Insertions AUG GGU AGG GAG GCA ACC UGA ACC GAC → AUG GGU AGG AGA GGC AAC CUG AAC CGA C ❖Deletions AUG GGU AGG GAG GCA ACC UGA ACC GAC → AUG GGU GGG AGG CAA CCU GAA CCG AC
37. GENETIC ENGINEERING ▪ Addition, deletion, or manipulation of a single trait in an organism to create a desired change ▪ Major tool: recombinant DNA
38. RECOMBINANT DNA • DNA generated by combination of two different DNA sources • Bacterial DNA + human gene for insulin production • Plant DNA + bacterial DNA → prevent disease for plant • Human gene + goat DNA → for blood clotting agent production
39. GENETIC ENGINEERING - BENEFITS ▪ Creates new crops and farm animals (crops grows in desert heat, or without fertilizer) ▪ Make bacteria that can make medicines, chemicals ▪ Grow human body parts ▪ Prevent genetic diseases
40. GEL ELECTOPHORESIS
41. Introducing free DNA into bacteria ❑Transformation- process of introducing free DNA into bacteria ▪ Competent cell- a cell that is capable of taking up DNA ▪ Heatshock ▪ Electroporation- use of an electric shock to momentarily open or disrupt cell walls ❑Conjugation- the contact of bacteria that involves the exchange of DNA with a mating tube ❑Transduction: injection of foreign DNA by a bacteriophage virus into the host bacterium
42. GENETICALLY MODIFIED ORGANISM (GMO) • Transgenic organisms (gmo) contain genes from other organisms • A clone: member of a population of genetically identical cells produced from a single cell
43. GEL ELECTROPHORESIS-forensic investigation 1. “Cut” DNA sample with restriction enzymes. 2. Run the DNA fragments through a gel. 3. Bands will form in the gel. 4. Everyone’s DNA bands are unique and can be used to identify a person. 5. DNA bands are like “genetic fingerprints”.
44. RESTRICTION ENZYME NOMENCLATURE ❑ Smith and Nathans (1973) proposed enzyme naming scheme ▪ Named for bacterial genus, species, strain, and type ▪ Three-letter acronym for each enzyme derived from the source organism ▪ First letter from genus ▪ Next two letters represent species ▪ Additional letter or number represents the strain or serotypes ❑ Ex: HindII was isolated from Haemophilus influenzae serotype d
45. RESTRICTION ENZYME
46. PROTECTION OF DNA ❑Why don’t bacteria destroy their own DNA with their restriction enzymes? • Bacteria protect their DNA from restriction digestion by methylation of its recognition site (by methylase) → prevents recognition by the restriction enzyme • Methylation is adding a methyl group (CH3) to DNA • Restriction enzymes are classified based on recognition sequence and methylation pattern
47. RESTRICTION ENZYME CLASSIFICATION ❑Type I • Multi-subunit proteins • Function as a single protein complex • Contain – two R (restriction) subunits – two M (methylation) subunits – one S (specificity) subunit • Cleave DNA at random length from recognition site
48. RESTRICTION ENZYME CLASSIFICATION ❑Type III • Large enzymes • Combination restriction-and-modification • Cleave outside of their recognition sequences • Require two recognition sequences in opposite orientations within the same DNA molecule • No commercial use or availability
49. STICKY END CUTTER HindIII EcoRI
50. USE OF RESTRICTION ENZYME RFLP analysis (Restriction Fragment Length Polymorphism) DNA sequencing DNA storage – libraries Transformation Large scale analysis – gene chips
51. ELECTROPHORESIS _ DNA is negatively charged from the phosphate backbone + Visualize DNA with ethidium bromide – fluoresces ONLY when bound to DNA
52. RESTRICTION ENZYME ▪ Digestion Condition • XbaI – Buffer 2: (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, pH 7.9 at 25°C.) – 100 μg/ml BSA – Incubate at 37° – 1 Unit digestS 1 μg DNA in 1 hour – Heat inactivate 65° for 20 min
53. RESTRICTION ENZYME ▪ Double Digest Enzyme Supplied NEBuffer % in B1 % in B2 % in B3 % in B4 SacI NEBuffer 1 + BSA 100 50 10 100 SacII NEBuffer 4 25 75 10 100 SalI NEBuffer 3 + BSA 0 0 100 0 SapI NEBuffer 4 75 50 0 100 Sau3AI NEBuffer 1 + BSA 100 50 10 100 Sau96I NEBuffer 4 50 100 100 100 SbfI NEBuffer 4 75 50 0 100 Double Digest Option 1. Mix the enzymes in the same compatible buffers 2. Conduct sequential digest Caution: some enzymes display star activity in certain buffers which causes them to digest the DNA at sites other than the standard recognition site.
54. CLONING VECTOR
55. DNA SEQUENCING
56. SANGER METHOD 2 3 1 4 Gel electrophoresis
57. RADIOACTIVELY LABELED ddNTPs ▪ Four different reactions are performed ▪ Each reaction contains either ddA, ddG, ddC, or ddT ▪ Autoradiography enables analysis of different fragment lengths corresponding to different termination points
58. Genetic Engineering Applications Genetic Engineering of Bacteria and Fungi a. Food Products b. Fermentation & Alcoholic Beverages c. Drugs, Vaccines, & Antibiotics d. Metabolism & Biofuel Production e. Human Gene Delivery Systems
59. Genetic Engineering Applications Genetic Engineering of Humans a. Germ Cell vs. Somatic Cell b. Gene Therapy c. Cloning, Stem Cells
60. GM BANANA ▪ Unique method of reproduction: asexual, not from seeds → the world may be soon out of bananas ▪ Fact: banana plantations in Africa, Asia and Central America: susceptible to fungi, viruses and pests → could be banana less in ten years → Copying genes of other fruits → instilling them into banana → disease resistance
61. GMO - Plant 1. Antioxidant –rich tomato 2. Nutrient –rich corn 3. Virus or herbicide – resistant crops 4. Virus-resistant Papaya 5. Drought or flood – resistant rice 6. Anti-malnutrition banana
62. FISHY STRAWBERRIES: strawberry resists frost damage 1. The flounder’s Idea: antifreeze gene is The flounder is a fish that live in icy copied and inserted seas. It has a gene that stops it from into a vector taken freezing to death. from a bacteria cell. Strawberries are soft fruits that can 2. The vector is put easily be damaged by frost. into a second bacterium. 3. This second bacterium is used to infect the strawberry cell. The flounder’s antifreeze gene enters the strawberry’s DNA. 4. GM strawberry plant can be bred many times. 5. GM strawberries make Strawberry a protein which helps cell with Strawberry that resists frost damage Antifreeze them resist frost. gene
63. TRANSGENIC ANIMAL How to Create a Transgenic Animal Designed DNA is added to an egg cell
64. GMF: PROS AND CONS Environment • "Friendly" bioherbicides and bioinsecticides • Conservation of soil, water, and energy • Better natural waste management • More efficient processing Society • More food for growing populations
65. Ethics • Violation of natural organisms' intrinsic values • Tampering with nature by mixing genes among species • Objections to consuming animal genes in plants and vice versa • Stress for animal Labeling • Not mandatory in some countries (e.g., U. States) • Mixing GM crops with non-GM confounds labeling attempts
66. Pros & Cons of GE Effects on the environment Pro arguments: Contra arguments: • GE minimizes • Every GMO released into soil erosion by the environment is a threat reducing the to the ecosystem because need of flowing. they are unpredictable by interacting with other living things in the environment, → it is difficult to assess • Plants resisant to the threats of GMO to the weather , climate ecosystem. insect infestation, desease, molds • GE can create toxins, and fungi. noxious-vegetation, harm to wild life and may create new molds and fungi.
67. Pros & Cons of GE Effects on the agriculture Pro arguments: Contra arguments: • Farmers can spray in • The weeds might order to kill weeds develop their spray resistance and greater without killing the crops. herbicide resistance has to be created. • The virus-resistance might also create new viruses that never existed before.
68. Bioremediation Biodegradation • Aerobic • Oxygen is reduced to water and the organic molecules (e.g. petroleum, sugar) are oxidized • Anaerobic • An inorganic compound is reduced and the organic molecules are oxidized (e.g. nitrate is reduced and sugar is oxidized) • NOTE: Many microbes can do both aerobic and anaerobic respiration; the process which produces the most ATP is used first!
69. Human Genome Project
70. February 2001 « Finished » sequence April 2003
71. Important features of Human Genome • 3 Billion chemical bases • The order of almost all (99.9%) nucleotide bases is exactly the same in all human individuals. • ~32,000 protein-coding genes • Average gene size is 3000 bp. Largest gene is dystrophin with 2.4 million bases. • About 1.5% of genes are coding for proteins
72. HUMAN CHROMOSOMES • 23 pairs of Chromosomes: a total of 46 • 22 pairs of autosomes or non sex chromosomes: a total of 44 • 1 pair of sex chromosomes: a total of 2
73. HUMAN GENOME ❑Human genome is nearly the same (99.9%) in all people ❑Only about 2% of the human genome contains genes which are the instructions for making proteins ❑Almost half of all human proteins share similarities with those of other organisms
74. What can we learn about genomes? • Within one genome: regulatory elements, gene order, chromatin structure • Through comparative studies: Evolution, conserved regions, rearrangements Gene quality and prediction.
75. NCBI Map Viewer
76. Ensembl Genome Browser
77. Applications to medicine and biology • Disease genes • Drug targets • Gene therapy
78. Using OMIM's Gene Map • What is Gene Map? With Gene Map, users can browse a table of genes organized by cytogenetic (di truyen te bao) map location • How do I search Gene Map? At the OMIM home page, select Search Gene Map from the blue navigation menu on the left. Search for all diseases on a specific chromosome, or for a specific disease
79. CURE CANCER WITH GENE THERAPY ▪ Gene p-53 normally keeps cells under control. ▪ The disease begins because the p-53 gene doesn’t work properly – perhaps because of a mistake in the gene code. ▪ Cure cancer by modifying faulty DNA to make the p-53 gene work. Lung cancer cells (530x)