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Last year’s headline @ CNN (9/20/2010)

Last year’s headline @ CNN (9/20/2010). In a heartbeat 15.59 % (2,167 votes) If the FDA approves it, I guess it's okay 10.45 % (1,453 votes) I'll wait a while and see how people fare 21.34 % (2,966 votes) In a pinch 4.99 % (693 votes) Not on your life 47.63 % (6,621 votes).

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Last year’s headline @ CNN (9/20/2010)

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  1. Last year’s headline @ CNN (9/20/2010) • In a heartbeat 15.59% (2,167 votes) • If the FDA approves it, I guess it's okay 10.45% (1,453 votes) • I'll wait a while and see how people fare 21.34% (2,966 votes) • In a pinch 4.99% (693 votes) • Not on your life 47.63% (6,621 votes) Clarified: What does "genetically modified" salmon mean? Public opinion poll: Would you eat genetically modified salmon? http://eatocracy.cnn.com/2010/09/20/genetically-modified-salmon/?hpt=T2 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  2. Chapter 2Technical Foundations of Genomics Recombinant-DNA techniques used in genomics © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  3. Contents • Introduction • Genomic and cDNA libraries • DNA Hybridization and Northern blots • Subcloning in vectors • Restriction-enzyme mapping • DNA sequencing • PCR amplification • Protein expression © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  4. What is the main goal of genomics? • Sequence the entire genome by cutting it into small, manageable pieces (fragments) • Assemble the entire genome from the pieces (fragments) • Make sense of the genome • Understand how gene expression takes place? • How life processes are networked? • Understand life?? © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  5. Introduction • Genomics built on recombinant-DNA technology (developed since early 1970s) • Thorough understanding of recombinant-DNA techniques • Prerequisite for understanding genomics technologies • Differences between genomics and recombinant-DNA technology • Genomics is high throughput approaches to allow more analyses in parallel • Genomics is dependent on computational analysis due to larger data sets © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  6. Genomic and cDNA libraries • Libraries are fragments of DNA cloned into a vector (microbial) but these are not organized according their natural arrangement on the chromosomes • Libraries are usually constructed before sequencing (prerequisite) • Genomic libraries are used for genomewide sequencing • cDNA libraries are needed for EST (expressed sequence tags) sequencing © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  7. Central Dogma Vector Genomic library DNA RNA Proteins Transcription Vector cDNA library cDNA mRNA Translation Expressed Sequence Tags ESTs © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  8. Genomic library • Made from fragments of genomic DNA • Genomic DNA cut up with restriction enzymes or randomly broken by mechanical shearing (passing through syringe needle, or by sonication) • Fragments ligated into cloning vectors • Small insert • Lambda phage: 20-50 Kbp • Plasmid: ~10 Kbp • Large insert • BACs (Bactetial Artificial Chromosomes) 100-300 kbp • YACs (Yeast Artificial Chromosomes) ~ 1 MBP © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  9. Restriction-enzyme mapping • Used for physical mapping of DNA • Restriction enzymes (RE) cut at defined sites • Palindromic sequences • Sites are landmarks on DNA • Then fragments are separated by gel electrophoresis CGATCG GTAC GCTAGC CATG Sticky end Versus blunt 3300 REs known but ~300 used 4 bp- 8 bp cutters GCGCGCGCGC CGCGCGCGCG © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  10. How to make a genomic library ori total genomic DNA ampR genomic DNA restriction enzyme ori anneal and ligate ampR ori plasmid (black) ori ori ampR ampR ampR same restriction enzyme transform E. coli; select for Amp resistance © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  11. Making a cDNA library • Step 1: Isolate RNA • RNA is purified from tissue or cell line • The mRNA is then isolated away from ribosomal and tRNAs • Column with oligo dT is used to bind poly A tissue or cell mRNA polyA stationary support polyT © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  12. Step 2: Obtain cDNA from RNA • mRNA is treated with the enzyme reverse transcriptase (RT) • The enzyme copies sequence of mRNA into first strand of DNA • Digest RNA with RnaseH • Another enzyme (RT) is used to make second strand of cDNA © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  13. Step 3: Transformation • Double-stranded cDNA is inserted into cloning vector • cDNA is ligated into cloning vector (plasmid or phage) • Vector is transformed or infected into bacteria plasmid E. Coli bacteria © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  14. Step 4: Library screening (both genomic and cDNA) A=T C=G selected colonies • Colony DNA is attached to membrane • DNA is screened with labeled probes • DNA is labeled with radioactivity • Labeled DNA is allowed to hybridize with DNA on membrane • After washing, positive hybridization spots are identified membrane Radioactive probe hybridization X-ray film © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  15. cDNA to EST cDNA library • For use in EST sequencing • Need to array individual clones • Library is spread on bacterial plates • Individual colonies are picked • Colonies are placed in test tubes or microtiter plates Clone 1 2 3 4 5 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  16. Colony picking • Automatic colony pickers play key role in genomics • Instead of manually picking one colony at a time, they identify and pick multiple colonies from plates • Pickers then deposit each colony into a microtiter well © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  17. Nucleic Acid Hybridization • Basis of microarrays for determining gene expression • Process by which complementary strands find each other • A–T and C–G base pairing • speed and fidelity: dependent on temperature, salt, sequence, and concentration (High temp and low salt) © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  18. Steps in Northern blotting © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  19. Northern blot • Gene expression analyzed by Northern blots • RNA samples undergo electrophoresis • RNA separated by molecular weight • Transferred to membrane • Probe labeled • Radioactivity or antibody ligand • Hybridized to RNA on membrane • Hybridization speed and fidelity dependent on time, temperature, salt concentration, and nucleic acid sequence and concentration © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  20. Northern blot example Time after elicitation • Example of time course of gene induction • Upper panel: RNA after electrophoresis (18S and 28S rRNA) • Bands correspond to ribosomal RNA (EtBR) • Probe detects two bands • Lower panel: Lower band shows rapid induction and then decline • Upper band shows slower induction, but stays induced for longer 0 2 4 6 8 10 12 24 M 0 2 4 6 8 10 12 24 – 4.2 kb – 2.1 kb © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  21. Northern blot and microarray 0 2 5 6 7 hrs 0 2 5 6 7 9 11 hrs DMC1 – SPS1 – DIT1 – SPS100 – DMC1 – SPS1 – 0 2 5 6 7 9 11 hrs fold repressed fold induced DIT1 – >20 10x 3x | 3x 10x >20 1:1 SPS100 – Identify genes whose expression was induced during sporulation in yeast © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  22. Cross-hybridization • Hybridization to a related, but not identical, sequence = cross-hybridization • Example: A probe from one member of a gene family is likely to hybridize to all other members • Problem in microarrays, particularly cDNA arrays • Oligonucleotide arrays prescreened to eliminate sequences likely to cross-hybridize © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  23. Subcloning • Propagating fragments of cloned DNA • Used for sequencing and protein production • Plasmid vectors • Replicate in bacteria • Resistant to antibiotics • Cloning sites ORI Region into which DNA can be inserted Plasmid cloning vector ampr © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  24. Subcloning: vector and fragment restriction enzymes • Vector and fragment to be inserted must have compatible ends • Sticky ends anneal • Enzyme ligase makes covalent bond between vector and fragment • Use of recombination instead of restriction sites DNA fragment cloning vector recombinant plasmid © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  25. Recombination cloning • Uses site-specific recombination for subcloning • DNA fragment flanked by recombination sites • Add recombinase “Clonase®” • Moves fragment from one vector to another © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  26. Transformation into bacteria • Bacteria prepared for transformation by making outer membrane permeable to DNA • Become competent • DNA added to bacteria • Heat shock (370 c) • Plate on selective media E. coli host cell recombinant plasmid transformed cell © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  27. Restriction-enzyme mapping • Used for physical mapping of DNA • Restriction enzymes cut at defined sites • Palindromic sequences • Sites are landmarks on DNA • Then fragments are separated by gel electrophoresis CGATCG GCTAGC © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  28. Gel electrophoresis • DNA fragments are separated by size in electric field • DNA negatively charged: proportional to size of fragment • Separated through gel matrix • Agarose or acrylamide © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  29. . . Log MW . . Distance Cutting a BAC with restriction enzymes • Separate DNA fragments are cut with restriction enzyme • DNA is visualized with ethidium bromide • Binds to DNA and fluoresces orange • The sizes of the fragments are determined based on a standard © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  30. DNA sequencing • Most current sequencing projects use the chain termination method • Also known as Sanger sequencing, after its inventor, Fredrick Sanger • Based on action of DNA polymerase • Adds nucleotides to complementary strand • Requires template DNA and primer © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  31. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  32. Chain terminates H dideoxyribonucleotide © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  33. Chain termination © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  34. Chain-termination sequencing • Dideoxynucleotides stop synthesis • Chain terminators • Included in amounts so as to terminate every time the base appears in the template • Use four reactions • One for each base: A,C,G, and T Template 3’ ATCGGTGCATAGCTTGT 5’ 5’ TAGCCACGTATCGAACA* 3’ 5’ TAGCCACGTATCGAA* 3’ 5’ TAGCCACGTATCGA* 3’ 5’ TAGCCACGTA* 3’ 5’ TAGCCA* 3’ 5’ TA* 3’ Sequence reaction products © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  35. Sequence separation – • Terminated chains need to be separated • Requires one-base-pair resolution • See difference between chains of X and X+1 base pairs • Gel electrophoresis • Very thin polyacryamide gel • High voltage • Works with radioactive or fluorescent labels + C A G T C A G T © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  36. A T C G Sequence reading of radioactively labeled reactions • Radioactive labeled reactions • Gel dried • Placed on X-ray film • Sequence read from bottom up • Each lane is a different base – + © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  37. Sequence detection • To detect products of sequencing reaction • Include labeled nucleotides • Formerly, radioactive labels were used • Now fluorescent labels • Use different fluorescent tag for each nucleotide • Can run all four reactions in same lane TAGCCACGTATCGAA* TAGCCACGTATC* TAGCCACG* TAGCCACGT* © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  38. Automated DNA sequencing © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  39. A T C G Assume all these lanes are merged in one but different colored bands – A=blue C= Green G= yellow T= red + Laser © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  40. Sequence reading of fluorescently labeled reactions • Fluorescently labeled reactions scanned by laser as particular point is passed • Color picked up by detector • Output sent directly to computer © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  41. Summary of chain termination sequencing © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  42. Polymerase chain reaction • Used in sequencing, diagnostics, comparative genomics, etc. • Uses thermostable DNA polymerase • Able to function near boiling temperature • Two primers complementary to sequences at 5’ and 3’ of region to be amplified • Double-stranded DNA template • Performed in thermal cyclers programmed to raise and lower temperature © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  43. PCR machines © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  44. PCR reaction: annealing primers • Template melted into two strands by high heat • > 90 degrees C • Primers anneal to both strands • Polymerase makes a copy of both strands © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  45. PCR reaction: amplification • Temperature raised to melt newly made DNA • Primers allowed to anneal as temperature drops • Polymerase elongates new second strand of DNA • Process repeated • Exponential increase in DNA © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  46. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  47. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  48. Protein expression • Important for proteomics • Need large amounts of recombinant protein for the following: • Structure determination • Antibody production • Protein arrays • Proteins made in bacteria, yeast, and insect cells • Then must purify the recombinant protein away from other proteins © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  49. Protein expression vectors • Protein expression vectors have the following: • Inducible promoters • Tags for purification • Histidines • Epitopes: FLAG or myc • Proteins: Maltose binding proteins • Coding sequence inserted in frame © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  50. Making recombinant protein • Expression vector transformed into bacteria • Bacteria grown to saturation • Compound added for induction • e.g., IPTG • Protein accumulates in bacteria © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

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