Bio115 29-31

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lukemlj
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Bio115 29-31
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2010-11-14 07:44:58
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Genetics lectures
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Genetics lectures 29, 30, 31
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  1. Base analogs
    chemicals with structures similar to any of the four standard bases
  2. 5-bromouracil
    base analog for thymine which can mispair with guanine leading to a transition mutation
  3. 2-aminopurine (2AP)
    base analog of adenine which can mispair with cytosine -> transition mutation
  4. EMS/Alkylation
    guanine->O6-Ethylguanine: pairs with thymine -> transition mutation
  5. Nitrous acid/Deamination
    cytosine->Uracil: pairs with adenine -> transition mutation
  6. Hydroxylamine/Hydroxylation
    cytosine->Hydroxylaminocytosine: pairs with adenine -> transition mutation
  7. oxidative radicals/reactions
    reactive forms of oxygen that damage DNA and induce mutations via chemical change
  8. intercalating agents
    molecules which insert into DNA in place of nitrogenous bases causing insertions and deletions, e.g. proflavin and acridine orange
  9. Hermann Muller
    1927: showed mutations in fruit flies inducible by radiation
  10. x-rays, gamma rays, cosmic rays
    alter base structure, break phosphodiester bonds, and even cause double-strand breaks
  11. UV light
    less energy, but still mutagenic
  12. pyrimidine dimers
    can be caused by UV light; create covalent bonds between bases which block replication
  13. SOS system
    eukaryotic system of eta polymerase that can bypass pyrimidine dimers
  14. Mismatch repair
    Replication errors, including mispaired bases and strand slippage
  15. Direct repair
    Pyrimidine dimers; other specific types of alterations
  16. Base excision
    Abnormal bases, modified bases, and pyrimidine dimers
  17. Nucleotide excision
    DNA damage that distors the double helix, including abnormal bases, modified bases, and pyrimidine dimers
  18. Mismatch repair Mechanism
    • Detects 3D distortion
    • cuts out distored part with exonuclease
    • fill in using original strand as template with DNA polymerase
    • repairs nicks with DNA ligase
    • Methylation at GATC sequence on old strand for differentiation
  19. exonuclease
    removes nucleotides usually at end of DNA strand
  20. DNA polymerase
    replaces nucleotides
  21. DNA ligase
    seals nick in sugar-phosphate backbone
  22. Direct Repair Mechanism
    • Converts modified nucleotides to original form
    • e.g. O6-Methyltransferase removes methyl group restoring base to guanine
    • e.g. photolyase uses light to break covalent bonds that link pyrimidine dimers
  23. photolyase
    an enzyme that uses light energy to break covalent bonds in pyrimidine dimers
  24. Base-excision repair mechanism
    • Excises modified bases and then replaces entire nucleotide
    • DNA glycosylase recognizes and removes damaged base
    • AP endonuclease cleaves phosphodiester bond on 5' site and removes sugar
    • DNA polymerase adds new nucleotide to 3'
    • DNA ligase fixes nick in sugar-phosphate backbone
  25. AP endonuclease
    removes nucleotide usually in middle of DNA strand
  26. Nucleotide-excision repair mechanism
    • Enzyme complex recognizes 3D distortion
    • DNA strand is separated and stabilized with binding proteins
    • An enzyme cleaves the strand on both sides of the damage
    • Part of damage strand is removed
    • Gap filled by DNA polymerase
    • Sealed by DNA ligase
  27. Homologous recombination
    A type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.
  28. Interstrand cross-link
    Two DNA strands are connected thru covalent bonds. Not much know about this.
  29. Common Mechanisms for Nucleotide removal
    • Detection
    • Excision
    • Polymerization
    • Ligation
  30. Detection
    Damaged section of DNA is recognized
  31. Excision
    DNA repair endonucleases nick phosphodiester backbone on one or both sides of the DNA damage and one or more nucleotides are removed
  32. Polymerization
    DNA polymerase addes nucleotides to the newly exposed 3'-OH group by using the other strand as a template and replacing the damaged nucleotides
  33. Ligation
    DNA ligase seals the nices in the sugar-phoshate backbone
  34. Differences in Mechanisms for Nucleotide removal
    How detection and excision are accomplished
  35. xeroderma pigmentosum
    autosomal recessive condition caused by nonfunctional repair mechanism for pyrimidine dimers
  36. 1973, Cohen and Boyer at UCSF
    Created first recombinant DNA molecule
  37. Recombinant DNA technology
    Set of molecular techniques for locating, isolating, altering, and studying DNA segments
  38. Stemps requiring Recombinant DNA techniques
    • Find gene
    • Separate gene
    • Make copies of gene
    • Insert gene into plasmid without degredation
    • Induce bacteria to take up plasmid
    • Select bacteria that take up plasmid
  39. Restriction enzymes/endonucleses
    • Enzymes that recognize and make double-strand cuts in DNA at specific nucleotide sequences
    • Produced naturally by bacteria to defend against viruses
  40. Type I and III Restriction enzymes
    Cut outside recognition sequence
  41. Type II restriction enzyme
    • Cuts within recognition sequence
    • Used in molecular genetic work
    • Names indicate original bacteria
    • More than 800 isolated
  42. Characteristics of restriction enzymes
    • Palindromic recognition sequence
    • Fragment end either cohesive or blunt
  43. Cohesive end restriction enzyme
    Staggered cut -> sticky ends
  44. Restriction Digest
    Reaction of mixture of DNA, buffer, restriction enzyme, water heated at about 37 C
  45. Electrophoresis
    Standard technique for separating molecules on basis of size/electrical charge
  46. agarose
    polysaccharide isolated from seaweed
  47. Viewing DNA fragments using electrophoresis
    DNA fragments move to positive pole with smaller fragments moving faster
  48. probe
    fluorescent or radioactive DNA or RNA fragment complementary to sequence of interest
  49. Southern blotting
    Transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by radioactive probe
  50. Northern blotting
    • A technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.
    • Size of mRNA molecule
    • Relative abundance of mRNA
    • Tissue in which MRNA is transcribed
  51. Western Blotting
    • Transfer of proteins from gel to a membrane
    • Probe is usually an antibody
    • Determine size of protein
    • Pattern of protein's expression
  52. Gene cloning
    create identical copies of a piece of DNA
  53. Cloning vector
    DNA molecule into which a foreign DNA fragment can be inserted for introduction into and replication in a cell
  54. Characteristics of an effective cloning vector
    • Origin of replication
    • selectable marker
    • one or more unique restriction sites where DNA can be inserted
  55. Types of cloning vectors
    • Plasmid, e.g. pUC19
    • Bacteriophage
    • Cosmid
    • BAC
    • YAC
    • Retroviral vectors
    • Transposons
    • Expression
  56. Transformation
    The capacity of bacterial cells to take up DNA from the environment
  57. Cosmid
    Plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection
  58. BAC
    Originally contructed from F plasmids
  59. YAC
    DNA molecule that has a yeast ORI, pair of telomeres, and a centromere
  60. Ti plasmid
    Plasmid that can be used to introduce DNA into plants
  61. Expression vector
    Vector that allows the production of protein (i.e. transcription and translation)
  62. PCR Steps
    • 1) Heat to 90-100 C for denaturation
    • 2) Cool to 30-65 C for primers to anneal
    • 3) Heat to 60-70 C for DNA synthesis
    • Repeat
  63. PCR Limitations
    • Requires knowledge of at least part of sequence for primers
    • Taq polymerase is poor at proofreading
    • Fragments larger than 50Kb cannot be isolated
  64. PCR as a diagnostic tool
    Detects presence of a particular sequence, e.g. HIV
  65. Shotgun cloning
    Clone all sequences in an organism into vectors
  66. DNA library
    Collection of clones containing all the DNA fragments from one source
  67. Genomic library
    Set of bacterial colonies or phages containing fragments in a DNA library
  68. cDNA library: isolating mRNA
    isolation of mRNA using oligo(dT) chains
  69. cDNA library: making cDNA from mRNA
    • oligo(dT) act as primers
    • reverse transcriptase for DNA strand
    • Rnase digests most of RNA strand
    • Remaining RNA act as primers for second DNA strand
  70. In situ hybridization
    A type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue
  71. Restriction Fragment Length Polymorphisms (RFLPs)
    Variations in the patterns of fragments produced when DNA is cut with a restriction enzyme typically caused by mutation
  72. Dideoxyribonucleoside triphosphate(ddNTP)
    No OH groups
  73. Sanger Method of DNA sequencing
    Uses ddNTP to terminate synthesis of strands of different length which can be read by electophoresis to sequence
  74. Pyrosequencing
    allows sequencing of entire genomes in a couple months
  75. DNA fingerprinting
    Indentification of individuals by short tandem repeats (STRs)

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