LE 20-2 Bacterium Bacterial chromosome Plasmid Gene inserted into plasmid Cell containing gene of interest Gene of interest DNA of chromosome Recombinant DNA (plasmid) Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Protein harvested Gene of interest Copies of gene Basic research on gene Basic research on protein Basic research and various applications Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth
LE 20-3 Restriction site DNA Restriction enzyme cuts the sugar-phosphate backbones at each arrow. One possible combination DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme DNA ligase seals the strands. Recombinant DNA molecule Sticky end
LE 20-7 Primers New nucleo- tides PCR – polymerase chain reaction. Making millions of copies of a portion of DNA to test. Thanks to bacteria from Yellowstone(with a heat stable DNA poly) – the reason forensics has evolved, sequencing the mammoth, Neanderthal etc.. We will do this on you!! 3 parts – heat DNA slightly to separate, cool so primers can bond, DNA poly adds nucleotides to 3” ends.
LE 20-8 Cathode Power source Anode Mixture of DNA molecules of differ- ent sizes Gel Glass plates Longer molecules Shorter molecules
LE 20-9 Normal -globin allele 175 bp201 bpLarge fragment Sickle-cell mutant -globin allele 376 bpLarge fragment Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles RFLP – restriction length polymorphisms. Mutations that can be detected to see if you have disease causing alleles.
Future Directions in Genomics Genomics is the study of entire genomes. Same gene can make different proteins by splicing introns at different places!! How do we know what a gene does? Knock it out and see what the consequences are. Gene targeting is often used to inactivate single genes. Such gene 'knockout' experiments have elucidated the roles of numerous genes in embryonic development, adult physiology, aging and disease. To date, more than ten thousand mouse genes (approximately half of the genes in the mammalian genome) have been knocked out. Ongoing international efforts will make 'knockout mice' for all genes available within the near future.
LE Cloned gene Retrovirus capsid Bone marrow cell from patient Inject engineered cells into patient. Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow