BOLOGY 107 Lecture 37Outline of Last Lecture I. Viruses a. HIVII. Viroids and Prionsa. Viroids b. PrionsOutline of Current Lecture III. Biotechnologya. DNA cloningb. Other DNA techniquesCurrent LectureBiotechnology1) DNA cloninga) Making multiple copies of DNAb) Recombinant DNAi) Combines DNA from many sourcesc) Plasmidsi) Circular, extra-chromosomal DNAii) Can b made to carry foreign DNAiii) Used for gene cloningd) Restriction enzymesi) Isolated from bacteria(1) Meant to protect from foreign DNA and viral infection(2) Cleaves DNA at specific sequences(a) “Restriction sites”(b) Leaves “sticky” sites with unique complementary sequences(3) Any restriction fragments with compatible ends can be glued together with ligasee) Process i) Isolate pieces of DNA(1) Cut with restriction enzymes(2) Separate by size on gel, cut up and purifyii) Ligate to form plasmidThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.iii) Transform into bacteriaiv) Grow lots of bacteria, make many copies of the plasmidf) Why do it?i) Subsequent cloning and DNA manipulationii) Have bacteria make a specific protein(1) Used for studies or drug productiong) Librariesi) Collection of different plasmids, viruses, or artificial chromosomes that together represent the whole genome of an individual(1) Can be genomic or cDNA(2) Stored in 96-well platesh) cDNAi) DNA copy of mRNA(1) RTase used to copy mRNA to DNA(2) DNA polymerase used to make copies(3) Can make a library using either the primer or just one specific mRNA2) Other DNA techniquesa) PCRi) Rapid and efficient DNA amplificationii) Used to copy specific DNAiii) Based on DNA replication(1) Uses Taq polymerase from Thermos aquaticus, a thermal Archaeaiv) Three steps repeated(1) Denature the DNA- separate with heat(2) Annealing- allow synthetic primers to bond while cooling(3) Extension- polymerase makes copies(4) Can be started at one sequence or done with the whole genomev) Uses(1) Diagnosis- detect virus or disease causing allele(2) Forensics- amplify DNA from evidence(3) Environmental monitoring- identify species in soil or water(4) Gene expression- measure changes in mRNA levels(5) Genetic manipulation- isolate for cDNA cloning(a) Evaluate mutation effect(b) Distinguish allelesvi) Problems(1) Taq polymerase has no proofreading ability(a) High error rate(2) It can also detect all DNA, including
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