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  1. 1. Human Gene Cloning and more molecular genetics ©2000-2006 Lee Bardwell Bio 97
  2. 2. Review Question A B ab 1 : 4 : 4 : 1 A b a B a b ©2006 Lee Bardwell A doubly-heterozygous Arrogant Beautiful fly (Nadia) was mated with a meek ugly testcross fly (Fred) AB Ab aB ab Are the genes linked ? (ignore statistics) If so, how far apart are they? Are Nadia’s recessive a and b alleles in cis or in trans? 1000 progeny were scored
  3. 3. lecture outline <ul><li>How can a clone (recombinant DNA molecule) of interest be found in a library? </li></ul><ul><li>How is DNA analyzed? </li></ul><ul><ul><li>purifying DNA </li></ul></ul><ul><ul><li>restriction digest </li></ul></ul><ul><ul><li>gel electrophoresis </li></ul></ul><ul><ul><li>visualizing DNA </li></ul></ul><ul><li>What’s a polymorphism? </li></ul><ul><ul><li>How is are used in positional cloning? (not in detail) </li></ul></ul><ul><ul><li>How are they used in forensics? </li></ul></ul>©2005 Lee Bardwell
  4. 4. Library <ul><li>If you clone restriction-digested human DNA into a plasmid, you get millions of plasmids, each containing a different piece of human DNA --> a library </li></ul><ul><li>We want to find a single book (e.g. a plasmid containing the insulin gene) in this library </li></ul>©2005 Lee Bardwell
  5. 5. MOVIE (http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf) Movie-steps in making a library (click here) http://highered.mcgraw-hill.com/sites/dl/free/0072437316/120060/ravenanimation.html From:
  6. 6. How to pick out your gene of interest ? <ul><li>Complementation - a gene on a plasmid can complement a mutant phenotype </li></ul><ul><ul><li>If cells express the phenotype - e.g. DNA repair defects </li></ul></ul><ul><li>Use an antibody (H&J p 368) </li></ul><ul><ul><li>Antibodies used to detect the bacterial colony expressing the protein of interest from the plasmid of interest </li></ul></ul>©1999 Lee Bardwell
  7. 7. More ways to pick out your gene of interest <ul><li>Hybridization (H&J p. 368) </li></ul><ul><ul><li>e.g. Cross-hybridization to related sequence </li></ul></ul><ul><ul><li>Hybridization to a marker (DNA sequence) near the gene of interest (see below) </li></ul></ul><ul><li>Positional cloning - find a marker that is near the gene using human gene mapping techniques </li></ul>©2005 Lee Bardwell
  8. 8. Purifying nucleic acid from cells <ul><li>Lyse the cells </li></ul><ul><li>Extract the proteins and lipids with organic solvent (e.g. phenol) </li></ul><ul><li>Precipitate the nucleic acid with ethanol </li></ul>©2000 Lee Bardwell
  9. 9. To determine the pattern of Restriction <ul><li>Enzyme cleavage in a DNA sample </li></ul><ul><li>Digest the DNA with the RE </li></ul><ul><ul><li>Purified DNA </li></ul></ul><ul><ul><li>Water </li></ul></ul><ul><ul><li>Buffer </li></ul></ul><ul><ul><li>RE </li></ul></ul><ul><li>2. Run the digested products on a gel to separate them by size </li></ul><ul><li>3. Stain the DNA in the gel (or do something else) so that you can visualize the DNA </li></ul>©2004 Lee Bardwell
  10. 10. Gel Electrophoresis <ul><li>Gel electrophoresis separates DNA molecules by size </li></ul><ul><li>An agarose gel is somewhat like jello </li></ul><ul><li>DNA migrates in an electric field </li></ul><ul><li>Larger molecules move slower due to agarose sieving effect </li></ul>©1999 Lee Bardwell
  11. 12. Visualizing DNA <ul><li>DNA bands on a gel can often be visualized by staining with dyes which bind DNA (ethidium bromide) </li></ul><ul><li>Methods used to detect very small amounts of DNA or to identify a single DNA band: </li></ul><ul><ul><li>Southern blot analysis </li></ul></ul><ul><ul><li>Polymerase chain reaction (PCR) </li></ul></ul>©1999 Lee Bardwell
  12. 14. 5 kb plasmid, 2 EcoRI sites E E plasmid Cut with EcoRI --> gel --> stain + (bottom) ©2000 Lee Bardwell - (top)
  13. 15. 5 kb plasmid, 2 EcoRI sites plasmid + (bottom) ©2006 Lee Bardwell - (top) E E Must cut 10 billion plasmid molecules to be able to see in gel
  14. 16. E E plasmid ©2005 Lee Bardwell H
  15. 17. Southern Blot Analysis <ul><li>A way to probe a gel containing a bunch of DNA fragments to try to identify a particular DNA fragment </li></ul>©2004 Lee Bardwell
  16. 18. Southern Blot Analysis - I <ul><li>DNA is cut into pieces by restriction enzymes </li></ul><ul><li>DNA fragments are separated by gel electrophoresis </li></ul><ul><li>DNA is transferred from gel to hybridization filter (blot) and denatured to produce single-stranded bands of DNA </li></ul>©2000 Lee Bardwell
  17. 19. H&J Fig. 6.27 modified Read H&J section 6.6 & 6.7
  18. 20. Southern Blot Analysis - II <ul><li>Filter is mixed with radiolabeled single-stranded DNA probe complementary to the DNA sequence of interest </li></ul><ul><li>hybridization = hydrogen bonds form between complementary base pairs </li></ul><ul><li>DNA bands hybridized to probe are detected by X-ray film exposure </li></ul>©1999 Lee Bardwell
  19. 21. H&J Fig. 6.27 modified Read H&J section 6.6 & 6.7
  20. 22. 5 kb plasmid, 2 EcoRI sites * * * * E E probe plasmid Cut with EcoRI --> gel --> blot Stained gel X-ray film ©2000 Lee Bardwell
  21. 23. GAATTC CTTAAG GAATTC CTTAAG Human chrm 9 Cut with EcoRI restriction enzyme Gel--> blot GAATTC CTTAAG probe * * * * 3 kb 2 kb X-ray film DNA sample ~ 750,000 other bands are not seen because they did not hybridize with the probe ©2000 Lee Bardwell
  22. 24. Polymerase Chain Reaction (PCR) <ul><li>Uses short (oligonucleotide) primers flanking the region of interest </li></ul><ul><li>Repeated rounds of DNA polymerization in an in vitro (test-tube) reaction result in the exponential amplification of the region of interest </li></ul><ul><li>(H&J section 6.7) </li></ul>©2000 Lee Bardwell REVIEW
  23. 25. Net Result… <ul><li>Of Southern blot or PCR </li></ul><ul><ul><li>detection one/few bands in a background of hundreds of thousands of bands </li></ul></ul>©2000 Lee Bardwell
  24. 26. Things human geneticists can’t do <ul><li>Establish true-breeding lab strains </li></ul><ul><li>Perform testcrosses or backcrosses </li></ul><ul><li>Score lots of progeny from the same mating </li></ul>©1999 Lee Bardwell
  25. 27. How to map a human disease gene <ul><li>Find a large, multigenerational, affected family </li></ul><ul><li>Test linkage of the disease to a mapped polymorphism </li></ul><ul><li>Do this for many, many markers until you find a closely linked one </li></ul>©1999 Lee Bardwell
  26. 28. We’re all different <ul><li>On average, any two people differ at 1 in every 1000 basepairs </li></ul><ul><li>Human vs. chimp... </li></ul>©2001 Lee Bardwell
  27. 29. Polymorphism Relatively common * genetic difference in a population ©1999 Lee Bardwell *Frequency of heterozygous genotypes is 10% or more
  28. 30. RFLPs polyms that alter the length of restriction fragments restriction fragment length polymorphisms <ul><li>Can result from… </li></ul><ul><ul><li>changes that introduce or delete an restriction enzyme site </li></ul></ul><ul><ul><li>differences in copy number of tandem DNA repeats </li></ul></ul>©2000 Lee Bardwell
  29. 31. H&J Fig. 4.19
  30. 32. H&J Fig. 4.20
  31. 33. Marker Cystic fibrosis gene Chrm 1 Chrm 7 50 cM { Closely linked marker linked marker Unlinked marker ©2000 Lee Bardwell A small (200-1000 bp) region of DNA from a known location on a given chrm
  32. 35. To look for linkage of a polymorphism to a disease gene <ul><li>Type each family member for the polym (=marker) and for the disease phenotype --> pedigree </li></ul><ul><li>A closely linked marker will segregate with the disease </li></ul>©1999 Lee Bardwell
  33. 36. Highly polymorphic markers * ... <ul><li>Are also useful at the scene of a crime </li></ul><ul><li>And in paternity cases </li></ul><ul><li>DNA typing, or “DNA fingerprinting” </li></ul><ul><li>(H&J Section 14.4) </li></ul>©2006 Lee Bardwell * Markers that can be used as probes to detect highly polymorphic regions
  34. 37. <ul><li>3 markers/probes </li></ul><ul><li>“ minisatellite” SSRs * </li></ul>DNA Fingerprinting *simple sequence repeats probe “a” b c a b c a b c a Probes Check out figures 14.18 and 14.19 ©2006 Lee Bardwell
  35. 38. H&J Fig. 14.17 . Allelic variation resulting from a variable number of units repeated in tandem in a nonessential region of a gene
  36. 39. Forensics Crime scene Sample: Victim + perp Victim AJ OJ DJ Suspects ©2000 Lee Bardwell
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