Trends in Biotechnology 110509 3b – Vectors

Chapter 3b - outline 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

Ch 3 b Learning Objectives 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.

DNA Cloning. There are several steps involved in cloning a gene in a cell. The specific steps in an individual procedure may vary, but most follow these steps: Isolation of DNA. Ligating the DNA into a vector. Transformation of a host cell with the recombinant DNA. Selection of host cells containing the recombinant DNA. Screening cells for those with recombinant DNA or producing a protein of interest.

Vectors We know that bacteriophage can transform bacteria. They do this by putting polynucleotides (DNA or RNA) into the bacteria. The bacteria then uses the information on the polynucleotide. The bacteria makes new bacteriophages.

There are other types of polynucleotides which can get into bacteria and other cells. These can also transform the cells. The cells will then make proteins and/or polynucleotides which are different from what the cell was making. The things which get the new polynucleotides into the cells are called vectors.

Genetic manipulation We can put pieces of DNA into the vectors. Then we can use the vectors to transform cells. The transformed cells then can produce the new DNA. The transformed cells may also express the proteins from the DNA.

Fig. 3.4 Ligation of a DNA fragment into a vector using the restriction endonuclease EcoR1 and DNA ligase.

Fig. 3.5 The transformation of bacteria and the selection of cells with the recombinant vector.

The recombinant DNA can be placed into a cell such as bacteria, and the bacteria will divide, creating cells called “clones.” Each clone will contain one or more identical copies of the recombinant DNA molecule.

One of the first experiments transferred some frog DNA into a bacterium. http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio38.swf::Early%20Genetic%20Engineering%20Experiment

Video: Key Steps of Molecular Cloning Other videos Video: Key Steps of Molecular Cloning http://www.youtube.com/watch?v=sjwNtQYLKeU&feature=related (20 Plasmid Cloning - Key Steps of Molecular Cloning.flv) Video: Plasmid Cloning http://www.youtube.com/watch?v=acKWdNj936o (20 Plasmid Cloning - Plasmid Cloning.flv)

We can also make better vectors. We can make vectors with many different restriction sites (regions of the vector which can be cut with restriction enzymes). We can also add genes which give resistance to different antibiotics. We can also add marker genes, or reporter genes. These are genes which show where the vector is, or show that it has been changed.

Cloning Vectors also need to: Have an origin of replication so that the DNA can be replicated within a host cell. Be small enough to be isolated without being degraded during purification.

pBR322 was one of the first plasmids created in the laboratory, and has the following characteristics: The molecule is small, and can be isolated easily. This vector can carry DNA of up to 5 to 10 kb. pBR322 has several unique restriction sites where the plasmid can be opened for inserting a DNA fragment. The genes for resistance to ampicillin (ampr) and tetracycline (tetr) are used for plasmid and DNA insert selection. A selectable marker: If one of the antibiotic resistance genes is broken, the bacteria will be sensitive to the antibiotic and die if treated with the antibiotic. A method that determines if a recombinant plasmid was created correctly and inserted correctly into the bacteria.

Fig. 3.6 Restriction map of pBR322 showing unique restriction sites, the ampicillin and tetracycline resistance genes, and the origin of replication.

Note how the cutting and insertion at a particular restriction site will result in loss of resistance.