1 / 49

Part Two – Lecture I

Part Two – Lecture I. Forms of DNA. A DNA. Rosalind Franklin focused on this form Prevalent under high salt concentrations More compact Modification of major and minor grooves. Z DNA discovered. 1979 – Andrew Wang – synthetic oligonucleotide 1.8 nm in diameter 12 base pairs per turn

Download Presentation

Part Two – Lecture I

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Part Two – Lecture I

  2. Forms of DNA

  3. A DNA • Rosalind Franklin focused on this form • Prevalent under high salt concentrations • More compact • Modification of major and minor grooves

  4. Z DNA discovered • 1979 – Andrew Wang – synthetic oligonucleotide • 1.8 nm in diameter • 12 base pairs per turn • G-C base pairs

  5. Ultracentrifugation and the Svedburg coefficient • DNA and RNA may be analyzed by ultracentrifugation • RNAs are differentiated according to their sedimentation behavior when centrifuged at high speeds in a concentration gradient

  6. Sedimentation Behavior • Sedimentation behavior depends upon the molecule’s • Density • Mass • Shape

  7. Sedimentation equilibrium centrifugation • A density gradient is created that overlaps the densities of the individual components of a mixture of molecules. • The gradient is usually made of a heavy metal salt such as CsCl • During centrifugation, the molecules migrate until they reach a point of neutral buoyant density

  8. Sedimentation equilibrium centrifugation • Can also be used to study the GC content • The number of GC pairs in the DNA molecule is proportional to the molecule’s buoyant density

  9. Denaturation and Renaturation of DNA Molecules • When denaturation of the double stranded DNA occurs, the hydrogen bonds open, the duplex unwinds, and the strand separate • No covalent bonds break so that the strands stay intact • Strand separation can be induced by heat

  10. Denaturation and uv spectrophotometry • Nucleic acids absorb ultraviolet light most strongly at wavelengths of 254-260 nm due to the interaction of the UV light and the rings of the purines and pyrimidines

  11. UV spectrophotometry • The increase of UV absorption of heated DNA is referred to as the hyperchromic shift and is easiest to measure

  12. Renaturation • Denaturation can be reversed – by slowly cooling the DNA • Single strands of DNA can randomly find their complementary strands and reassociate • The hydrogen bonds will form slowly and then more and more duplexes or double helixes will form

  13. Molecular Hybridization • This technique is based upon the denaturation and renaturation of DNA • In this case DNA from two different sources can be mixed • DNA and RNA and be mixed together – a transcript can find its complementary sequence in DNA

  14. Molecular Hybridization • Used to determine the amount of complementarity or similarity between two different species

  15. Proteins are polymers • Proteins are polymers of amino acids. They are molecules with diverse structures and functions. • Polymers are made up of units called monomers • The monomers in proteins are the 20 amino acids

  16. Blotting Procedures

  17. Autoradiograph

  18. Fluorescent in situ hybridization - FISH • In this procedure mitotic or interphase cells are fixed to slides and subjected to hybridization conditions. • Biotin is complexed with the DNA and then bound to a fluorescent molecule such as fluorescein

  19. Examples of fluorescence

  20. Reassociation kinetics - Britten • Used with small fragments of DNA • DNA is then denatured • Temperature is lowered and reassociation monitored • Used to compare different organisms • Originally uncovered repetitive DNA sequences due to a greater than anticipated complmentarity

  21. Reassociation kinetics and repetitive DNA

  22. Electrophoresis • Separates molecules ina mixture by causing them to migrate under the influence of an electric field • A sample is placed in a porous media such as agarose or polyacrylamide gel • They are then placed in a solution (buffer) which conducts an electric current

  23. Separation of DNA • DNA has a strong negative charge due to the phosphate groups • When the DNA is placed in the gel, it will migrate toward the positive electrode

  24. Agarose Gel Electrophoresis

  25. Staining

  26. SDS Polyacrylamide Gels • Vertical gel • SDS used to denature proteins • Proteins run or separate according to their molecular mass

  27. Native Protein Gels

  28. Native Gels • In native gels, the proteins migrate according to a mass/charge ratio • In the case of hemoglobin the variant forms are able to be separated based upon a difference of charge due to the substitution of amino acids from the Beta globin chain

  29. Protein Facts • Proteins: Polymers of Amino Acids • Proteins are polymers of amino acids. They are molecules with diverse structures and functions. • Each different type of protein has a characteristic amino acid composition and order. • Proteins range in size from a few amino acids to thousands of them. • Folding is crucial to the function of a protein and is influenced largely by the sequence of amino acids.

  30. Proteins: Polymers of Amino Acids • Each different type of protein has a characteristic amino acid composition and order. • Proteins range in size from a few amino acids to thousands of them. • Folding is crucial to the function of a protein and is influenced largely by the sequence of amino acids.

  31. Proteins are complex molecules • They have levels of structure • Structure based upon the sequence of the amino acids

  32. Polar side chains

  33. Non Polar Hydrophobic side chains

  34. Electrical charged hydrophilic

  35. Function of Proteins - continued • Enzymes – Biological catalysts • Transport of small molecules – Albumin and haptoglobin • Transport of oxygen – hemoglobin and myoglobin • Membrane proteins – to assist in support • Channels in membranes – to allow the passage of molecules or ions • Electron carriers in electron transport in the production of ATP

  36. Functions( continued)i • Clotting proteins • Immune proteins to fight infectious agents • Histones – DNA binding proteins • Toxins to repel or kill other organisms • Bacteriocins – molecules produced by bacteria against bacteria

  37. Functions of proteins • Hormones – Growth hormone • Receptors – to Receive information so that cell can communicate with other cells • Neurotransmitters – messenger molecules – to send information between neurons • Cytoskeleton – actin, myosin, and collagen – the structure of connective tissue and muscles • Antibodies – Immunoglobulins to fight disease

  38. Four levels of Protein Structure • There are four levels of protein structure: primary, secondary, tertiary, and quaternary. • The precise sequence of amino acids is called its primary structure. • The peptide backbone consists of repeating units of atoms: N—C—C—N—C—C. • Enormous numbers of different proteins are possible.

  39. The causes of Tertiary structure

More Related