1. Ch 20: DNA Technology

2. The mapping and sequencing of the human genome has been made possible by advances in DNA technology. Human genome project We are now developing techniques for making recombinant DNA Genes from two different sources - often different species - are combined into the same molecule. DNA technology has launched a revolution in biotechnology. The manipulation of organisms or their components to make useful products. DNA technology is now applied in areas ranging from agriculture to criminal law, but its most important achievements are in basic research. Introduction

3. Recombinant DNA – Taking DNA from two sources and combining then into one molecule. –Occurs naturally in viral transduction, bacterial transformation, and conjugation Biotechnology (genetic engineering) –Engineering genes in the Lab Recombinant DNA

4. Many tools and techniques have been developed to manipulate and engineer genes. 1.Restriction Enzymes 2.Gel Electrophoresis -Restriction Fragment Polymorphisms 3.DNA Probe 4.Polymerase Chain Reaction 5.Complementary DNA

5. Restriction Enzymes Discovered in the 1960s Extracted from bacteria –Used to fend off bacteriophages –Appear to serve a host-defense role –Protect own DNA by methaylation of adenines & cytosines REs cut DNA at specific sites called recognition sequences or sites. –Leaving fragments of DNA Scientists have isolated 100s of REs –Named for the bacteria in which they were found. EcoRI BamHI HindIII clip

6. Naming Restriction enzymes are named based on the bacteria in which they are isolated in the following manner: EEscherichia(genus) cocoli(species) RRY13(strain) IFirst identifiedOrder ID'd in bacterium

7. EnzymeOrganism from which derived Target sequence (cut at *) 5' -->3' Ava IAnabaena variabilisC* C/T C G A/G G Bam HIBacillus amyloliquefaciensG* G A T C C Bgl IIBacillus globigiiA* G A T C T Eco RIEscherichia coli RY 13G* A A T T C Eco RIIEscherichia coli R245* C C A/T G G Hae IIIHaemophilus aegyptiusG G * C C Hha IHaemophilus haemolyticusG C G * C Hind IIIHaemophilus inflenzae RdA* A G C T T Hpa IHaemophilus parainflenzaeG T T * A A C Kpn IKlebsiella pneumoniaeG G T A C * C Mbo IMoraxella bovis*G A T C Mbo IMoraxella bovis*G A T C Pst IProvidencia stuartiiC T G C A * G Sma ISerratia marcescensC C C * G G G SstIStreptomyces stanfordG A G C T * C

8. The restriction sites are often a symmetrical series of four to eight bases on both strands running in opposite directions. If the restriction site on one strand is 3’- CTTAAG-5’, the complementary strand is 5’- GAATTC-3’. Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts. Copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a specific enzyme. CLIP

9. Restriction enzymes cut covalent phosphodiester bonds of both strands often in a staggered way creating single-stranded ends, sticky ends. These extensions will form hydrogen-bonded base pairs with complementary single- stranded stretches on other DNA molecules cut with the same restriction enzyme. These DNA fusions can be made permanent by DNA ligase which seals the strand by catalyzing the formation of phosphodiester bonds.

10. Must use same restriction enzyme on both.

12. Setting up a simple restriction digestion: 1.DNA: Reliable cleavage by restriction enzymes requires DNA that is free from contaminants such as phenol or ethanol. Excessive salt will also interfere with digestion by many enzymes, although some are more tolerant of that problem. preferences for ionic strength 2.An appropriate buffer: Different enzymes cut optimally in different buffer systems, due to differing preferences for ionic strength and major cation. When you purchase an enzyme, the company almost invariably sends along the matching buffer as a 10X concentrate. 3.The restriction enzyme!

13.  Restriction Enzyme animation Restriction Enzyme animation  Animation #2#2  Cloning a Gene Cloning a Gene Bacteria DNA that has DNA from another organism spliced in to it.

14. Gel Electrophoresis Method of rapidly analyzing and comparing genomes.

15. Restriction Fragment Length Polymorphisms (RFLPs) The restriction pattern is different for every organism. This is why you get different banding patterns. Gel Electrophoresis

16. DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye intercalates between bases of DNA and RNA Agar Agar is an unbranched polysaccharide obtained from the cell walls of some species of red algae or seaweed

17. Rate of movement depends on size, electrical charge, and other physical properties of the macromolecules.

18. Separation depends mainly on size (length of fragment) with longer fragments migrating less along the gel.

19. Simulation Because DNA has a negative charge, it moves toward the opposite side. Smaller fragments move greater distances clip Animation

21. We start by adding the restriction enzyme to each of the three samples to produce restriction fragments. We then separate the fragments by gel electrophoresis. Southern blotting (Southern hybridization) allows us to transfer the DNA fragments from the gel to a sheet of nitrocellulose paper, still separated by size. enhancing the result of an agarose gel electrophoresis by marking specific DNA sequencesSouthern blotting -method in molecular biology of enhancing the result of an agarose gel electrophoresis by marking specific DNA sequences This also denatures the DNA fragments. Bathing this sheet in a solution containing our probe allows the probe to attach by base-pairing (hybridize) to the DNA sequence of interest and we can visualize bands containing the label with autoradiography. We can tie together several molecular techniques to compare DNA samples from three individuals Animation

22. Common method of creating copies of specific fragments of DNA PCR rapidly amplifies a single DNA molecule into many billions of molecules Polymerase Chain Reaction (PCR); Making copies Animation

23. Complementary DNA ( c DNA) introns present a problemWhen scientists clone a human gene in a bacterium, the introns present a problem. Bacteria lack introns and have no way to cut them out. Animation

24. In order to clone a human gene in a bacterium, the introns need to be removed. The genes is allowed to be transcribed and fully processed into mRNA. Reverse transcriptase is added to the mRNA and DNA copies are made. The DNA made by this process is called cDNA. Complementary DNA ( c DNA)

25. DNA probe; DNA Tagging A DNA probe is a radioactively labeled single strand of nucleic acid molecule used to tag a specific sequence in a DNA sample. The probe bonds to a complementary sequence wherever it occurs. Can identify genetic defects Animation

26. Gene Therapy

27. Cloning

28. Genetically Modified Organisms Clip Transgeneic Organisms CLIPCLIP

29. Transgenic Organisms72 CLIP Transgenic Tobacco, from 1986. This is an ordinary photographic image of a tobacco plant engineered to express a firefly gene which produces luciferase.

30. Plasmids as vectors Lab on Fri Animation

31. Golden Rice 23 times more Vitamin A Called a Transgenic Organism71

32. Researchers use recombinant DNA technology to analyze genetic changes. They cut, splice together, and insert the modified DNA molecules from different species into bacteria or another type of cell that rapidly replicates and divides. The cells copy the foreign DNA right along with their own DNA. An example of this is the gene for human insulin inserted into a bacterium. This is how human insulin is mass produced.

33. Not only does genetic engineering have applications in medicine and the environment, it also has uses in industry and agriculture. Sheep are used in the production of alpha- 1 antitrypsin, which is used in the treatment of emphysema. Goats are also producing the CFTR protein used in the treatment of cystic fibrosis.

34. In the plant world, the buds of cotton plants are vulnerable to worm attacks. The buds of a modified cotton plant resist these worms, resulting in increased cotton production. These gene insertions are ecologically safer than pesticides. They affect only the targeted pest.

35. Plant biologists have used DNA technology to produce plants with many desirable traits. These include increased disease resistance, herbicide resistance, and increased nutritional content.

36.  Scientists today have developed genetically altered bacteria.  Among them are strains of bacteria that  eat up oil spills  manufacture alcohol and other chemicals  process minerals.  There is concern about possible risks to the environment and the general population as genetically engineered bacteria are introduced.