Lecture 7 - Genome Tools

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Site-directed mutagenesis •Theoretically, can modify protein at protein-level or gene-level •Protein-level: chemical, post-translational, modifications are harsh, nonspecific and tedious •But, mirrors Post-translational and epigenomic •Structure and predictive tools necessary for gene-level •“Directed mutagenesis” -generate amino acid coding changes at DNA level
Oligonucleotide-directed mutagenesis with M13 DNA •Best case scenario, let Nature do the work •Known sequence; available clone •Clone into ssM13 vector •Mismatch with mutation of desire •DNA Pol-based synthesis •Tf E. coli with ds recombinant vector •Expect 50%; in practice, 1-5% recovery •Why?
Page 1 and 2: Mammals as bioreactor •Use of ani
Page 3 and 4: •http://www.conncoll.edu/ccacad/z
Page 5 and 6: •http://www.chinadaily.com.cn/chi
Page 7 and 8: “…missed it, by this much”
Page 9 and 10: •Stem cells
Page 11 and 12: •http://en.wikipedia.org/wiki/Emb
Page 13 and 14: •http://en.wikipedia.org/wiki/Emb
Page 15 and 16: Irving Weissman: hemapoietic stem c
Page 17 and 18: •http://en.wikipedia.org/wiki/Amg
Page 19 and 20: •http://en.wikipedia.org/wiki/Amg
Page 21 and 22: •http://en.wikipedia.org/wiki/Epo
Page 23 and 24: •http://en.wikipedia.org/wiki/Epo
Page 26 and 27: •http://en.wikipedia.org/wiki/Hem
Page 28 and 29: •http://en.wikipedia.org/wiki/Hem
Page 30 and 31: •http://en.wikipedia.org/wiki/Ima
Page 32 and 33: Lecture 6: Chapter 8 •Directed mu
Page 34 and 35: Natural selection and growth: How?
Page 36 and 37: Natural mutation: HBB, beta hemoglo
Page 38 and 39: Another perspective: people and mut
Page 42 and 43: Tools: deamination repair systems
Page 44 and 45: Enrichment for mutations: dut/ung
Page 46 and 47: Variation: Directed mutagenesis wit
Page 48 and 49: Second generation variation: Random
Page 50 and 51: Random mutagenesis with error-prone
Page 52 and 53: Hybrids generated by shuffling prot
Page 54 and 55: Mutant proteins with non-universal
Page 56 and 57: Protein engineering •2003 •1000
Page 58 and 59: Reasons to mutate: One immediate go
Page 60 and 61: Example 2: ribonuclease •Bull sem
Page 62 and 63: Changing Asn to other AAc •At hig
Page 64 and 65: Increasing enzymatic activity •Mo
Page 66 and 67: Metal cofactor requirement •Modif
Page 68 and 69: Decreasing protein sensitivity •S
Page 70 and 71: Modifying protein specificity •Pr
Page 72 and 73: Modifying antibodies •Hypervariab
Page 74 and 75: Modifying two properties: Increasin
Page 76 and 77: Altering multiple properties simult
Page 78 and 79: Laundry, detergent and mushrooms
Page 80: Molecular analysis of hybrid peroxi
Page 83 and 84: Therapeutic agents •Horse/cow ser
Page 85 and 86: Bioprospecting: Microbes with desir
Page 87 and 88: Aspirin: “Age of Industrializatio
Page 89 and 90: Natural Products: Tamoxifin •Tamo
Page 91 and 92:
Natural Products: Bioprospecting
Page 93 and 94:
Recombinant proteins for human use
Page 95 and 96:
Hybrid products: INF •IFN cDNA is
Page 97 and 98:
Recombinant modification: hTNF-α
Page 99 and 100:
Optimizing gene expression •Multi
Page 101 and 102:
Treatment with secreting bacteria
Page 103 and 104:
Treatment •Genentech: hDNase I in
Page 105 and 106:
Clearing the lungs 2 with alginate
Page 107 and 108:
Alginate lyase[s] •ORF 69,000 Da
Page 109 and 110:
Phenylketonuria (PKU) •Autosomal
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Lecture 7 - Genome Tools

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