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1. Announcements Online Quiz #15 – Deadline Tonight True or False Recombinant DNA technology is the manipulation and combination of DNA molecules from different sources.

2. Recombinant DNA and Biotechnology

3. Recombinant DNA and Biotechnology Cleaving and Rejoining DNA Getting New Genes into Cells Sources of Genes for Cloning Some Additional Tools for DNA Manipulation Biotechnology: Applications of DNA Manipulation

4. Application of Recombinant DNA Recombinant DNA technology is the manipulation and combination of DNA molecules from different sources. http://www.pharmaceutical-technology .com/projects/eli_lilly/images/eli3.jpg http://www.abpischools.org.uk/resourc es04/pharm_business/images/fig14.jpg https://www2.vaxserve.com/views/images/photo/8315-01.jpg

5. Humulin & Insulin Humulin recombinant human insulin: a form of insulin (trade name Humulin) made from recombinant DNA that is identical to human insulin; used to treat diabetics who are allergic to preparations made from beef or pork insulin - wordnet.princeton.edu/perl/webwn A polypeptide hormone secreted by the islets of Langerhans and functioning in the regulation of the metabolism of carbohydrates and fats, especially the conversion of glucose to glycogen, which lowers the blood glucose level. Any of various pharmaceutical preparations containing this hormone that are derived from the pancreas of certain animals or produced through genetic engineering and are used in the medical treatment and management of diabetes mellitus (type I). - http://www.answers.com/topic/insulin

6. Tools of Biotechnology Tools of Biotechnology Restriction Endonucleases Vectors Gene Libraries Gel Electrophoresis Polymerase Chain Reaction DNA Sequencing Reporter Genes Southern Hybridization …and many more tools.

7. Figure 16.1 Bacteria Fight Invading Viruses with Restriction Enzymes

8. Cleaving and Rejoining DNA Bacteria defend themselves against invasion by viruses by producing restriction enzymes which catalyze the cleavage of DNA into small fragments. The enzymes cut the bonds between the 3 hydroxyl of one nucleotide, and the 5 phosphate of the next. There are many such enzymes, each of which recognizes and cuts a specific sequence of bases, called a recognition sequence or restriction site (commonly 4 to 6 base pairs long). EcoRI E: Escherichia co: coli R: strain RY13 I: first restriction enzyme isolated from E. coli http://en.wikipedia.org/wiki/I mage:Restriction_enzyme.jpg

9. Cleaving and Rejoining DNA Host DNA is not damaged due to methylation of certain bases at the restriction sites; this is performed by enzymes called methylases. The enzyme EcoRI cuts 5 ... GAATTC ... 3 3... CTTAAG ... 5 Notice that the sequence is palindromic: It reads the same in the 5-to-3 direction on both strands. http://www.sci.osaka-u.ac.jp/introduction/eng/images/b_17.gif

10. EcoRI http://en.wikipedia.org/wiki/Image:EcoRI.png

11. EcoR1 in Action http://juang.bst.ntu.edu.tw/BCbasics/images/EcoRI.gif

12. Sticky End & Blunt Ends http://www.langara.bc.ca/biology/mario/Assets/ResEnzyme.jpg

13. Cleaving and Rejoining DNA What good are they? Restriction enzymes serve as “knives” for genetic surgery. Hundreds of restriction enzymes with different recognition sequences have been purified from various organisms, so the knives come in many forms. Problem: DNA cut with restriction enzymes creates a collection of fragments all within a microcentrifuge tube. Typically, we want only one of the fragments. Solution: Gel Electrophoresis http://www.scidynamics.com/images/EasyFit-tube.jpg

14. Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

15. Cleaving and Rejoining DNA The fragments of DNA can be separated using gel electrophoresis. Because of its phosphate groups, DNA is negatively charged at neutral pH. When DNA is placed in a semisolid gel and an electric field is applied, the DNA molecules migrate toward the positive pole. Smaller molecules can migrate more quickly through the porous gel than larger ones. After a fixed time, the separated molecules can then be stained with a fluorescent dye and examined under ultraviolet light.

16. Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)

17. Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)

18. Gel Electrophoresis Electrophoresis gives two types of information: Size of the DNA fragments can be determined by comparison to DNA fragments of known size added to the gel as a reference. http://www.nippongene.com/pages/products/ electrophoresis/marker/onestep/osl500_1000.jpg http://www-biology.ucsd.edu/classes/bicd101.SP06/Gel.jpg

19. Southern Hybridization And… A specific DNA sequence can be determined by using a complementary labeled single-stranded DNA probe. Modified http://www.ansi.okstate.edu/ research/2004rr/25/25_files/image004.gif

20. Figure 16.3 Analyzing DNA Fragments

21. Cleaving and Rejoining DNA SPLICING DNA Simple techniques give scientists the power to manipulate genetic material; this has revolutionized biological science in the past 30 years. If two different DNAs are cut so each has sticky ends, fragments with complementary sticky ends can be recombined and sealed with the enzyme DNA ligase. http://www.slic2.wsu.edu:82/hurlbert/micro101/images/LigaseAnimation6.gif

22. Gene Splicing http://www.scholarnet.co.nz/member/courses/sol/data/images/biology/gene_splicing.gif

23. Figure 16.4 Cutting and Splicing DNA

24. Getting New Genes into Cells Plasmids are ideal vectors for the introduction of recombinant DNA into bacteria. http://www.biologie.uni-regensburg.de/Zoologie/Schneuwly/Explab/4eco.gif

25. Getting New Genes into Cells A plasmid is small and can divide separately from the host’s chromosome. They often have just one restriction site, if any, for a given restriction enzyme. Cutting the plasmid at one site makes it a linear molecule with sticky ends. If another DNA is cut with the same enzyme, it is possible to insert the DNA into the plasmid. Plasmids often contain antibiotic resistance genes, which serve as genetic markers.

26. Figure 16.5 (a) Vectors for Carrying DNA into Cells

27. Inserting a Gene

29. Getting New Genes into Cells Only about 10,000 base pairs can be inserted into plasmid DNA, so for most eukaryotic genes a vector that accommodates larger DNA inserts is needed. For inserting larger DNA sequences, viruses are often used as vectors. If the genes that cause death and lysis in E. coli are eliminated, the bacteriophage  can still infect the host and inject its DNA. The deleted 20,000 base pairs can be replaced by DNA from another organism, creating recombinant viral DNA.

30. Getting New Genes into Cells Bacterial plasmids are not good vectors for yeast hosts because prokaryotic and eukaryotic DNA sequences use different origins of replication. A yeast artificial chromosome, or YAC, has been made that has a yeast origin of replication, a centromere sequence, and telomeres, making it a true eukaryotic chromosome. YACs have been engineered to include specialized single restriction sites and selectable markers. YACs can accommodate up to 1.5 million base pairs of inserted DNA.

31. Figure 16.5 (b) Vectors for Carrying DNA into Cells

32. Getting New Genes into Cells Plasmid vectors for plants include a plasmid found in the Agrobacterium tumefaciens bacterium, which causes the tumor-producing disease, crown gall, in plants. Part of the tumor-inducing (Ti) plasmid of A. tumefaciens is T DNA, a transposon, which inserts copies of itself into the host chromosomes. If T DNA is replaced with the new DNA, the plasmid no longer produces tumors, but the transposon still can be inserted into the host cell’s chromosomes. The plant cells containing the new DNA can be used to generate transgenic plants.

33. Figure 16.5 (c) Vectors for Carrying DNA into Cells

34. Getting New Genes into Cells When a population of host cells is treated to introduce DNA, just a fraction actually incorporate and express it. In addition, only a few vectors that move into cells actually contain the new DNA sequence. Therefore, a method for selecting for transfected cells and screening for inserts is needed. A commonly used approach to this problem is illustrated using E. coli as hosts, and a plasmid vector with genes for resistance to two antibiotics.

35. Figure 16.6 Marking Recombinant DNA by Inactivating a Gene

36. Getting New Genes into Cells Other methods have since been developed for screening. The gene for luciferase, the enzyme that makes fireflies glow in the dark, has been used as a reporter gene. Green fluorescent protein, which is the product of a jellyfish gene, glows without any required substrate. Cells with this gene in the plasmid grow on ampicillin and glow when exposed to ultraviolet light.

37. Sources of Genes for Cloning Gene libraries contain fragments of DNA from an organism’s genome. Restriction enzymes are used to break chromosomes into fragments, which are inserted into vectors and taken up by host cells.

38. Figure 16.7 Construction of a Gene Library

39. Sources of Genes for Cloning Using plasmids for insertion of DNA, about one million separate fragments are required for the human genome library. Phage , which carries four times as much DNA as a plasmid, is used to hold these random fragments. It takes about 250,000 different phage to ensure a copy of every sequence. This number seems large, but just one growth plate can hold as many as 80,000 phage colonies.

40. Sources of Genes for Cloning A smaller DNA library can be made from complementary DNA (cDNA). Oligo dT primer is added to mRNA tissue where it hybridizes with the poly A tail of the mRNA molecule. Reverse transcriptase, an enzyme that uses an RNA template to synthesize a DNA–RNA hybrid, is then added. The resulting DNA is complementary to the RNA and is called cDNA. DNA polymerase can be used to synthesize a DNA strand that is complementary to the cDNA.

41. Figure 16.8 Synthesizing Complementary DNA

42. Sources of Genes for Cloning If the amino acid sequence of a protein is known, it is possible to synthesize a DNA that can code for the protein. Using the knowledge of the genetic code and known amino acid sequences, the most likely base sequence for the gene may be found. Often sequences are added to this sequence to promote expression of the protein. Human insulin has been manufactured using this approach.

43. Sources of Genes for Cloning With synthetic DNA, mutations can be created and studied. Additions, deletions, and base-pair substitutions can be manipulated and tracked. The functional importance of certain amino acid sequences can be studied. The signals that mark proteins for passage through the ER membrane were discovered by site-directed mutagenesis.

44. Some Additional Tools for DNA Manipulation Homologous recombination is used to study the role of a gene at the level of the organism. In a knockout experiment, a gene inside a cell is replaced with an inactivated gene to determine the inactivated gene’s effect. This technique is important in determining the roles of genes during development.

45. Figure 16.9 Making a Knockout Mouse (Part 1)

46. Figure 16.9 Making a Knockout Mouse (Part 2)

47. Some Additional Tools for DNA Manipulation The emerging science of genomics has to contend with two difficulties: The large number of genes in eukaryotic genomes The distinctive pattern of gene expression in different tissues at different times To find these patterns, DNA sequences have to be arranged in an array on some solid support. DNA chip technology provides these large arrays of sequences for hybridization.

48. Figure 16.10 DNA on a Chip

49. Some Additional Tools for DNA Manipulation Analysis of cellular mRNA using DNA chips: In a process called RT-PCR, cellular mRNA is isolated and incubated with reverse transcriptase (RT) to make complementary DNA (cDNA). The cDNA is amplified by PCR prior to hybridization. The amplified cDNA is coupled to a fluorescent dye and then hybridized to the chip. A scanner detects glowing spots on the array. The combinations of these spots differ with different types of cells or different physiological states.

50. Some Additional Tools for DNA Manipulation DNA chip technology can be used to detect genetic variants and to diagnose human genetic diseases. Instead of sequencing the entire gene, it is possible to make a chip with 20-nucleotide fragments including every possible mutant sequence. Hybridizing that sequence with a person’s DNA may reveal whether any of the DNA hybridized to a mutant sequence on the chip.