Trends in Biotechnology Chapter 3a – Tools for Recombinant DNA

Chapter 3 - outline Cutting and Joining DNA Separating Restriction Fragments and Visualizing DNA DNA Cloning V. Cloning Vectors A. Bacterial Vectors 1. Plasmids 2. Bacteriophage 3. Cosmids B. Vectors for Other Organisms 1. Yeast Artificial Chromosomes (YACs) 2. Bacterial Artificial Chromosomes (BACs) 3. Plant Cloning Vectors 4. Mammalian Cell Vectors

VI. Cell Transformation VII. Constructing and Screening a DNA Library A. Genomic Library B. cDNA Library C. Screening Libraries D. Expression Libraries VIII. Reporter Genes

Southern Blot Hybridization Northern Blot Hybridization Polymerase Chain Reaction DNA Sequencing XIII. Protein Methods A. Protein Gel Electrophoresis B. Protein Engineering C. Protein Sequencing XIV. DNA Microarray Technology A. Biotech Revolution: RNA Interference Technology: Gene Silencing XV. Applications of Recombinant DNA Technology

Learning Objectives 1. Know how restriction endonucleases work to cut DNA, and why they are important in biotechnology. Compare blunt ends with sticky ends. 2. Know how electrophoresis separates pieces of DNA. 3. List and know the steps of DNA cloning.

4. Know how vectors are used to transform bacteria, and how to select for successfully transformed bacteria. Compare the how different vectors can carry different sizes of DNA into the bacteria. 5. List the types of vectors that can be used to transform yeast, mammalian cells and plants, and why they are effective in those organisms. 6. List the methods of transformation of cells.

7. Compare genomic libraries, cDNA libraries, and expression libraries 7. Compare genomic libraries, cDNA libraries, and expression libraries. How are they constructed? What are the libraries looking for? How they are screened? 8. List the various types of reporter genes used in research. 9. Compare Northern and Southern blot hybridization. How are they constructed? What is each type of hybridization looking for?

10. Know the function of PCR, the steps of PCR, and what researchers do with PCR. 11. Compare the two methods of DNA sequencing: the chemical method and the Sanger method, and know which method is more widely used. How does automation change DNA sequencing?

12. List and define the various methods of analyzing proteins 12. List and define the various methods of analyzing proteins. Are any of these methods similar to DNA methods? 13. Know the types of microarrays, and how DNA and protein microarrays work. 14. List the applications of recombinant DNA technology.

Fig. 3.1 Cutting double-stranded DNA. (a) The enzyme DNase cuts DNA at random sites.

Fig. 3.1 (b) Restriciton enzymes cut DNA at specific sites.

Figure 3 a: Restriction enzymes make double-stranded cuts in the sugar-phosphate backbone of DNA http://www.nature.com/scitable/resource?action=showFullImageForTopic&imgSrc=44956/pierce_18_2_FULL.jpg

Video: Restriction http://www.dnalc.org/resources/animations/restriction.html (13 restriction.exe)

Fig. 3.1 (c) A Specific restriction enzyme cuts each DNA molecular at the same sequence site.

Fig. 3.2 The ligation of two different pieces of DNA.

Figure 3 b: Restriction enzymes producing cohesive, or sticky, ends. These ends can be joined again. http://www.nature.com/scitable/resource?action=showFullImageForTopic&imgSrc=44956/pierce_18_2_FULL.jpg

Video: restriction enzymes bio37 http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf (13 restriction)

Fig. 3.3 Agarose gel electrophoresis is used to separate DNA (and RNA) molecules according to size.

Electrophoresis: http://www.dnalc.org/resources/animations/gelelectrophoresis.html (20 Electrophoresis - gelelectrophoresis.exe) Together, restriction enzymes and gel electrophoresis can give a lot of information. http://www.dnalc.org/view/16529-Animation-24-The-RNA-message-is-sometimes-edited-.html (20 Electrophoresis - rest enzy and electro.exe)