BIOLCHEM 415 1st Edition Lecture 38Outline of Last Lecture I. Protein synthesis decodes the information in mRNAII. Peptidyltransferase catalyzes peptide-bond synthesisIII. Bacteria and eukaryotes differ in the initiation of protein synthesisIV. A variety of biomolecules can inhibit protein synthesisV. Protein synthesis is regulated by a number of mechanismsOutline of Current Lecture VI. Recombinant DNA techniques are very important for biotechnology and medicineVII. Restriction enzymes cut out DNA sequences that need to be amplifiedCurrent LectureBiotechnology and medicine- recombinant DNA- identification, diagnosis, treatment- e.g. recombinant human insulinRestriction enzymes - found in prokaryotes - palindromic target sequences- axis of symmetry (both strands cut)- act as primitive immune system in bacteria- only cleaves foreign DNABlotting techniques - southern : DNA (template) – DNA probe hybridized to DNA 1 – transfer DNA to membraneThese 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.2 - hybridize membrane with radiolabeled probe (annealing)3 – wash and autoradiograph- northern : RNA (template) – DNA probe hybridized to RNA - western : protein – antibody probe binds to specific proteinSequencing of DNA - need a template and primer and dNTPs, polymerase, dideoxys- dideoxynucleotides lack 3’ OH and cause chain termination Fluorescent detection of oligonucleotide fragments - dideoxy sequencing methodCloning DNA fragments- vehicle for replication of DNA called a vector - plasmid, bacteriophage, virus, whole genome- a vector is capable of replication - DNA to be cloned is ligated to vector- amplified with vector in host cellComplementary DNA (cDNA) generated by reverse transcriptase- generated from mRNA template- retroviral enzyme- RNA template, primer, dNTPs, divalent metal ions (Mg2+)- cDNA inserted into cloning vector to generate cDNA libraryMolecular cloning of a gene1 – cut plasmid with restriction enzyme 2 – cut DNA to be cloned with compatible restriction enzymes 3 – ligated using DNA ligase4 – transform bacteria5 – grow bacteria and isolate plasmidSolid phase synthesis of DNA chain (synthetic oligonucleotides )Taq DNA polymerase- commonly used enzyme in PCRPCR- synthesize enzymatically amplified DNA in a test tube- sensitive- yield = 2n (n = # of cycles)- quantitative- tell you how highly expressed a gene is- in presence of fluorescent dyeOligonucleotide – directed mutagenesis- produce desired change in DNA sequenceRNA interference- disrupt gene expression – synthetic siRNAs- RISC complex- RNAi = potential therapeutic approach- delivery and safety methods must be worked outTransgenic mice- carry foreign genes as part of own genomeGene disruption by homologous recombination- replication, repair (diversity)Sequencing of Human Genome- finished sequence reported in 2004- ~ 21000 genes that encode proteinsHigh throughput/next gen/deep sequence technology- sequence fragments of DNA from many
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