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Molecular Technologies

Molecular Technologies. Discovery of DNA structure/function (1953) Molecular Biology Powerful Techniques - Gene Analysis & Manipulation Widely Applicable Ecology, Behavior, Medicine, Agriculture Pharmaceuticals, Horticulture, Animal Breeding, etc. 1. Denaturation of DNA.

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Molecular Technologies

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  1. Molecular Technologies Discovery of DNA structure/function (1953) Molecular Biology Powerful Techniques - Gene Analysis & Manipulation Widely Applicable Ecology, Behavior, Medicine, Agriculture Pharmaceuticals, Horticulture, Animal Breeding, etc.... 1

  2. Denaturation of DNA High temperature, high pH - break H bonds hyperchromatic effect 2

  3. Denaturation of DNA Relative G:C content 3

  4. Renaturation of DNA Homology affects renaturaton (hybridization) Stringency [Salt]Temperature High Low High Low High Low 4

  5. Renaturation of DNA Concentration affects rate of hybridization 5

  6. Renaturation of DNA Concentration affects rate of hybridization 6

  7. Nucleic Acid Probes DNA/RNA probes Target sequences 7

  8. Phylogenetic Relationships Amount bound ~ homology 8

  9. In situ Hybridization FISH 9

  10. In situ Hybridization Chromosome Painting 10

  11. In situ Hybridization Detecting Chromosomal Rearrangements 11

  12. One specific sequence at each spot Hybridize with labeled RNA from different cells or individuals (red or green) Detect binding and interpret results 12

  13. Microarrays (Gene Chips) Demonstration: http://www.bio.davidson.edu/Courses/genomics/chip/chip.html http://learn.genetics.utah.edu/content/labs/microarray/ 13

  14. Microarrays (Gene Chips) mRNA isolated from cells (cancer/normal; aerobic/anaerobic) Converted to cDNA Fluorescent labels http://www.bio.davidson.edu/Courses/genomics/chip/chip.html 14

  15. Microarrays (Gene Chips) Mixture of labeled cDNA hybridized to microarray Specific binding Scan and evaluate expression http://www.bio.davidson.edu/Courses/genomics/chip/chip.html 15

  16. Microarrays (Gene Chips) 16

  17. Microarrays (Gene Chips) Applications: Evaluate gene expression Development, Cancer, Disease progression Determine presence of specific mutations Genotyping, SNP association studies Detect infectious genomes Virus, Bacteria, etc. 17

  18. Restriction Endonucleases Bacterial immune system Recognize and cleave foreign DNA Names EcoRI - E. coli strain RI HinDIII - H. influenzae strain DIII Each recognizes and cuts a specific sequence of DNA Specific molecular scissors 18

  19. Restriction Endonucleases Palindromic Sequence ‘AND MADAM DNA’ 5’>3’ = 3’>5’ 19

  20. Restriction Endonucleases Ends: Blunt or Sticky (complementary) 3’ or 5’ overhangs 20

  21. Restriction Endonucleases Recognition Methylation Enzyme Site Sensitive ? AluI 5’...AG CT...3’ No 3’...TC GA...5’ BamHI 5’...G GATCC...3’ No 3’...CCTAG G...5’ BspEI 5’...T CCGGA...3’ Yes 3’...AGGCC T...5’ KpnI 5’...GGTAC C...3’ No 3’...C CATGG...5’ 21

  22. Recombinant DNA RE fragments from two sources joined by DNA ligase 22

  23. Cloning DNA DNA inserted into vector for growth in another cell Vector enables growth in host cell (ori or ARS) Selective markers (antibiotic resistance, nutritional requirement) 23

  24. Plasmid Vector Bacterial Vector pUC19 pBluescript LacZ at MCS 24

  25. Eukaryotic Vectors Yeast Artificial Chromosome Shuttle Vectors CEN; ARS plus ORI; markers 25

  26. Gene Cloning Source of DNA Insert Genomic DNA - dsDNA - coding and noncoding cDNA - RNA > DNA - coding only One specific gene or Comprehensive collection - Genomic Library, cDNA Library 26

  27. Genomic Library Clone collection of overlapping fragments (contigs) Ave. fragment size 256 bp = (44) 4096 bp = (46) Comprehensive library for 3 x 109 bp genome 4096 bp fragments > 732,000 clones 27

  28. Genomic Library Probability of having at least one copy of any DNA sequence in a genomic library N = ln(1-P) N = number of clones needed ln(1-f) P = probability desired f = ave. size fragment cloned/genome size Ex. Human genome 106 kb; if fragments average 15 kb, More than 920,000 clones are needed 28

  29. cDNA Library (Expression Library) cDNA - complementary DNA copy mRNA - reverse transcriptase, polydT primer 29

  30. Transferring Recombinant DNA into Host Cells Transformation Each colony contains vector, Only some have insert Clones - each with specific segment of genome 30

  31. Identifying Transformants Containing Gene Inserts Blue/White Screen - pBluescript Ampicillin resistant lacZ gene -galactosidase IPTG Xgal (white) > blue White colonies all contain insert in MCS 31

  32. Screening Clones for Specific Gene Sequences Detection of specific sequence - probe Colonies transferred to membrane DNA released, hybridization with probe Identify clone with desired gene 32

  33. Screening cDNA Libraries for Specific Gene Products Detection of protein produced by cells with desired gene Specific Antibody Identify clone expressing gene 33

  34. Screening Clones for Ability to Rescue Mutants DNA genomic library in shuttle vector used to transform eukaryotic cells with defective gene Only those with good copy of the gene grow Permissive (30OC) Restrictive (37OC) Complementation 34

  35. Screening Clones for Ability to Rescue Mutants Cloning of yeast ARG1 gene 35

  36. Restriction Mapping Relative positions of RE sites along DNA - molecular markers BstEB bp distances 670 1,500 500 250 36

  37. Restriction Mapping Specificity of enzymes - reproducible fragments produced 37

  38. Determining Sizes of Restriction Fragments Gel Electrophoresis - Agarose (200 bp+), Polyacrylamide Determine distance migrated * Extrapolate size * 38

  39. Determining Sizes of Restriction Fragments Visualize DNA bands - Ethidium Bromide, UV transilluminator 39

  40. Generating Restriction Maps kb kb kb kb kb Answer: 4 H 7 E 9 Complete Digestion with HindIII and/or EcoRI 40

  41. Generating Restriction Maps Partial Digestion with PstI Possible partial digestion fragments (*complete) 19, 15, 14, 12, 10, 8*, 7, 5*, 4*, 2* kb Is a 6 kb fragment possible? A 13 kb fragment? 41

  42. Generating Restriction Maps Partial Digestion with SalI Which fragments are adjacent? 4.2 = 3.5 = 8.2 = 42

  43. Using Restriction Maps to Analyze Constructs Characterize insert in recombinant DNA of clone 43

  44. Using Restriction Maps to Analyze Constructs Orientation of insert can be determined 44

  45. Analysis of Genomic Sequences Human genome - cut with RE (6 bp site) - ~7.3 X 105 fragments How can you find and identify a specific fragment? 45

  46. Southern Blot Analysis Southern Blot DNA transfer - detect fragment specific probe Northern Blot RNA Western Blot Protein Transfer to nylon membrane Hybridize with probe Visualize autoradiography chemiluminescence 46

  47. RFLP Analysis Restriction Fragment Length Polymorphisms (RFLP) Basis for polymorphic DNA sequences 1. Change in RE cleavage site Gel Pattern A a 47

  48. RFLP Analysis Southern blot with probe to repeat Basis for polymorphic DNA sequences 2. Change in number of repeats VNTR (variable numbers tandem repeats: 10 - 1000 bp) STRP (simple tandem repeats: CAGCAGCAG) 48

  49. SNP Analysis using Southern Blot Single Nucleotide Polymorphisms that affect restriction sites 49

  50. Mutation Detection using Southern Blot Detection of Sickle-cell gene by DdeI fragment 50

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