DNA-Based Information Technologies

1. DNA-Based Information Technologies • DNA Cloning: The Basics • From Genes to Genomes • From Genomes to Proteomes • Genome Applications and New Products of Biotechnology

3. Paul Berg Stanley N. Cohen & Herbert Boyer, 1970s (p.1119) DNA Cloning: (recombinant DNA technology or genetic engineering) 1. Cutting DNA at precise location by restriction endonucleases. 2. Joining two DNA fragments by DNA ligase. 3. Selecting a small molecule of DNA capable of self-replication. DNA segment can be joined to cloning vectors (plasmids or viral DNA) to form recombinant DNA. 4. Moving recombinant DNA from the test tube to a host cell for replication. 5. Selecting or identifying host cells that contain recombinant DNA.

4. Restriction Endonucleases and DNA Ligase Yield Recombinant DNA.

6. Restriction endonucleases: • type II (cut at recognition sites) • type I (cut at >1000 bp away) & type III (cut at 25 bp away) • also contain methylase activities

7. WRONG! Not by EcoRV!!! Dimeric EcoRV??? sticky end Mg2+

8. Restriction digestion: EcoR1 cut sticky ends PvuII cut blunt ends

9. New DNA sequences can be created by inserting synthetic DNA fragment (linkers) between the ends that are being ligated. An insert with multiple restriction sites is called a polylinker.

10. Terminal transferase can be used to generate sticky ends for joining two DNA fragments.

11. Cloning Vectors Allow Amplification of Inserted DNA Segments pBR322

12. Cloning foreign DNA in E. coli with pBR322 (plasmid): small fragment Transformation by CaCl2, 0oC > 42oC or by electroporation Positive clones: ampR gene disrupted by insert

13. Cloning foreign DNA in E. coli with bacteriophage l: ~40 kbp fragment

14. Cloning foreign DNA with bacterial artificial chromosomes (BACs): large fragment Positive clones: lacZ gene disrupted by insert

15. Recombinant DNA Technology • DNA Cloning: The Basics • From Genes to Genomes • From Genomes to Proteomes • Genome Applications and New Products of Biotechnology

16. DNA libraries provide specialized catalogs of genetic information • Genomic library • cDNA library

17. Contig: ordering of the clones in a DNA library Sequence-tagged site (STS) can provide landmarks for genomic sequencing projects.

18. Isolating a Gene from a Cellular Chromosome Cloning a gene often requires a DNA library constructing a cDNA library: (complementary DNA) Expressed sequence tag (EST): Partial sequences of cDNA library at random useful in the mapping of large genomes.

19. Specialized cDNA library: fusing cDNAs to a marker or reporter gene Example 1: green fluorescence protein (GFP)

20. Example 2: epitope tag

21. Specific DNA sequence can be amplified e.g., by PCR (polymerase chain reaction): Kary Mullis

22. DNA amplified by PCR can be cloned

23. Hybridization allows the detection of specific sequences probe (i.e., labeled DNA or RNA) is complementary to the DNA being sought

24. The Southern blot procedure, as applied to DNA fingerprinting.

25. Designing a probe to detect the gene for a protein of known amino acid sequence. All 8 will match at least 17 of 20 positions.

26. Genome sequences provide the ultimate genetic libraries Human Genome Project Strategy:

27. Human Genome Project, started at late 1980 by 20 centers of six nations (coordinated by NIH/USA), led first by Watson and after 1992 by Collins. The completed sequence of the human genome (3x109 bp) was published in April 2003 (efforts spanning 14 yrs). Joining by Celera Co. (funded in 1997 by Venter) accelerated the process (two years ahead of schedule). James D. Watson

28. Genomic sequencing timeline

29. Only <1.4% of our DNA acturally encodes proteins

30. Recombinant DNA Technology • DNA Cloning: The Basics • From Genes to Genomes • From Genomes to Proteomes • Genome Applications and New Products of Biotechnology

31. Proteome: the complement of proteins expressed by a genome. Proteomics: a field of investigation evolved from the concept of proteome. Protein functions: Phenotypic function: the effect of a protein on the organism. Cellular function: the network of interactions engaged in by a protein at the cellular level. Molecular function: the precise biochemical activity of a protein.

32. Comparative genomics: Sequence or structural relationships provide information on protein function Conserved gene order (synteny) in the mouse and human genomes

33. Cellular expression patterns can reveal • the cellular function of a gene. • Methods to detect cellular expression patterns: • Two-dimensional gel electrophoresis • DNA microarrays (DNA chips) • Protein chips

34. Photolithography • Ways to make a DNA chip: • Spot synthesized DNA fragment • (nanoliter), by robotic devices, • onto a solid surface of the chip. • Direct synthesize DNA fragment by • programmed computer, joining one • nucleotide to the next in a • photoreaction (photolithography), • on the solid surface of the chip. >>

35. DNA microarrays provide compact libraries for studying genes and their expression.

36. Enlarged image of a DNA chip: 6200 genes of the yeast genome

37. Detection of protein-protein interactions helps • to define cellular and molecular function • Comparisons of genome composition (Fig.9.24) • Purification of protein complexes (IP by Ab x tag) • Yeast two-hybrid analysis (Fig.9.25)

38. Use of comparative genomics to identify functional genes: (Proteins P3 and P6 may be functionally related)

39. The yeast two-hybrid system

41. Recombinant DNA Technology • DNA Cloning: The Basics • From Genes to Genomes • From Genomes to Proteomes • Genome Applications and New Products of Biotechnology

42. Application of Recombinant DNA Technology Cloned genes can be expressed using expression vector.

43. Cloned genes can be altered: e.g., by site-directed mutagenesis Michael Smith

44. Yeast is an important eukaryotic host for recombinant DNA: Very large DNA segments can be cloned in yeast artificial chromosomes (YACs). up to 2 x 106 bp genomic fragments isolated by pulse field electrophoresis

45. Cloning DNA in plant system: aided by bacterial plant parasites

46. Cloning in Plants Is Aided by a Bacterial Plant Parasite Agrobacterium tumefaciens which contains the large (~200 kbp) Ti plasmid. Transfer of plasmid to host chromosome relies on the 25 bp repeats and the vir gene products (of Ti plasmid). The vir gene is inducible by the phenolic compound acetosyringone (released by wounded plant cell).

47. Metabolites produced in Agrobacterium-infected plant cells by T DNA encoded enzymes, that benefit the bacterium and form a plant tumor. unusual a.a. growth hormones

48. A two-plasmid strategy to create a recombinant plant.

49. A tobacco plant in which the gene for firefly luciferase is expressed

50. Tomato plants engineered to be resistant to some insect larvae (right) that express protein (by bacterium Bacillus thuringiensis) toxic to moth larvae.