L6 Vectors Presentation PDF

Vectors Requirements for a cloning vector a) Should be capable of replicating in host cell b) Should have convenient RE sites for inserting DNA of interest c) Should have a selectable marker to indicate which host cells received recombinant DNA molecule. Exampl...

Vectors Requirements for a cloning vector a) Should be capable of replicating in host cell b) Should have convenient RE sites for inserting DNA of interest c) Should have a selectable marker to indicate which host cells received recombinant DNA molecule. Example:LacZ gene is used as a screening marker. LacZ gene produces the enzyme β-galactosidase which breaks down lactose to glucose and galactose d) Should be small and easy to isolate  Types of vectors 1. Bacterial plasmids are small, circular DNA molecules that are separate from the bacterial chromosome. They replicate independently of the bacterial chromosome. Useful for cloning DNA inserts less that 20 kb (kilobase pairs). Inserts larger than 20 kb are lost easily in the bacterial cell.  2. Bacteriophage lambda (45 kb) contains a central region of 15 kb that is not required for replication or formation of progeny phage in E. coli. Thus, lambda (a bacterial virus) can be used as a cloning vector by replacing the central 15 kb with 10-15 kb of foreign DNA. This is done as follows: mix RE cut donor DNA and lambda DNA in test tube, ligate, use in vitro packaging mix that will assemble progeny phage carrying the foreign DNA, infect E. coli with the phage to amplify  3. Cosmids are hybrids of phages and plasmids that can carry DNA fragments up to 45 kb. They can replicate like plasmids but can be packaged like phage lambda. 4. Expression vectors are vectors that carry host signals that facilitate the transcription and translation of an inserted gene. They are very useful for expressing eukaryotic genes in bacteria.  Making a Library Libraries are collection of DNA clones in a certain vector. The goal is to have each gene represented in the library at least once.  Types of libraries according to the source of donor DNA a) Genomic - made from RE DNA fragments of total genomic DNA b) Chromosome – made from RE DNA fragments of one chromosome isolated via flow cytometry or pulsed field gel electrophoresis c) cDNA (complementary DNA) – made from DNA synthesized from mRNA  Probing for a gene in the library DNA probes are based on the fact that a denatured (heated or chemically treated to become single stranded) DNA molecule will hybridize (bind) to sequences that match or are similar to it.  Where does the probe DNA come from? (1) cDNA from highly expressed mRNA from a tissue (2) homologous gene from a related organism (3) DNA obtained from “reverse genetics” (protein to DNA): If you have the protein product of the gene in which you are interested….. sequence part of the protein, synthesize a short (>20 nucleotides) DNA probe based the protein sequence using the genetic code and use as your probe.  DNA Vaccines With DNA vaccines, the subject is not injected with the antigen but with DNA encoding the antigen. The DNA is incorporated in a plasmid containing: 1. DNA sequences encoding one or more protein antigens or, often, simply epitopes of the complete antigen(s).  2. DNA sequences incorporating a promoter that will enable the DNA to be efficiently transcribed in the human cells. 3. Sometimes DNA sequences encoding – Co stimulatory molecules – Sequences that target the expressed protein to specific intracellular locations (e.g., endoplasmic reticulum) are included as well. Recombinant plasmid  The DNA vaccine can be injected into a muscle just as conventional vaccines are. In contrast to conventional vaccines, DNA vaccines elicit cell- mediated — as well as antibody-mediated — immune responses.  The cell-mediated response The plasmid is taken up by an antigen presenting cells(APC). The gene(s) encoding the various components are transcribed and translated. The protein products are degraded into peptides. These are exposed at the cell surface nestled in class I histocompatibility molecules where they serve as a powerful stimulant for the development of cell mediated immunity.  The antibody-mediated response If the plasmid is taken up by other cells (e.g. muscle cells), the proteins synthesized are released and can be engulfed by antigen- presenting cells (including B Cells). In this case, the proteins are degraded in the class II pathway and presented to helper T cells. These secrete lymphokines that aid B cells to produce antibodies.  Some recombinant DNA products being used in human therapy Insulin for diabetes Factor VIII for males suffering hemophilia A Factor IX for hemophilia B Human growth factor Erythropoietin for anemia Parathyroid hormone Hepatitis B surface antigen for vaccination