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BIOL 205
Ian D Chin- Sang

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BIOL 205:week 9-10, lecture 26-27 Recombinant DNA What is needed? • a tool to cut DNAinto pieces (enzymes: specific pieces or mechanical breakage: random pieces) • a means of identifying and purifying DNApieces of interest! • enzymes : to make and manipulate DNA • a means to replicate DNA(so we have lots of DNAto work with). Eukaryotes • almost all mRNA is made a precursor first → direct copy of DNA • then, is processed: 5' cap and polyA-tail, and pieces cut out • same for rRNAand tRNA: all made as large precursors then modified ◦ tRNA: capture specific amino acids that recognize codons • looking at genomic DNA: won't resemble mRNAthat codes protein What types of DNAcan we work with ? Histone mRNAdoesn't have poly-Atail Circles backon itselfonce reaches end:and can use this end to make 2 strand Choose polymerase based on length need to synthesize (pol3thousands ofnts, pol120-50nts) Have to know sequence before hand What does a gene look like in a eukaryote/mammalian genome Can be thousands to 100 thousand nts long 5' cap protected: exonucleases don't recognize it (5' to 5') Why transcribe to much RNA w/ junk? -timing -regulate when mature mRNA is expressed - ex:in early development in womb → cell adhesion molecule in Drosophila involved in neuronal guidance - just because have intron, doesn't mean it is used • Drosophila DSCAM gene for neuronal guidance • contains blocks of highly similar exons, each mRNA picks up one each • if all possible combinations are expressed then there would be 38.016 different proteins Enzymes used in molecular genetics 1. restriction enzymes- cut DNAinto defined segments 2. DNAligase – join fragments of DNA 3. DNApolymerases- synthesize DNAon a template (if want mutations find one that doesn't have a proofreading function) 4. reverse transcriptase – copy RNA into DNA 5. nucleases – deoxyribonucleases and ribonucleases • exonuclease –cleaves from the ends 5’or 3’ • endonuclease – cleaves internally 6. RNApolymerase- make RNAon a template 7. terminal transferase – add a nucleotide to either 5' or 3' 8. polynucleotide kinase – add a phosphate 9. alkaline phosphate – remove a phosphate Restriction Enzymes • cleave DNAin a sequence-specific manner! • evolved as a defense mechanism---protect against the invasion of foreign DNA • different bacteria make different restriction enzymes Anynon-methylated DNAwillbe cleaved (E. colimethylates allofITS DNA, so anythingthat isn't is foreign) Many restriction sites recognize palindromic sequences • palindromes: Same sequence read forward as backward • in English : kayak, rotator, radar, “Able was I ere I saw Elba” end up w/ 5' overhang when have overhang can hybridize to sequence that has complementary overhang If sequences were random (if DNAwas random, which it is not) the distribution frequency would be: • 4 base cutter = 4 = 1 cut every 256 bp 6 • 6 base cutter= 4 = 1 cut every 4096 bp • 8 base cutter= 4 = 1 cut every 65,536 Cut DNA can be separated by agarose or polyacrylamide gel electrophoresis DNA = negatively -smaller charged fragments travel furtherthan largerones not a carcinogen Formation of a recombinant DNA molecule Whenhave pieces ofDNA want a vector:to replicate DNAso have lots and lots of pieces Insert is usually3xas muchas vector preventingvector from closingback together w/ out insert - but stillwillhave some small percentage ofreformed vecot plasmid Puttinginsalt solutionand incubating Vector Features • must contain a replicon that enables it to eplicate in host cells (region of DNAthat is amplified, i.e.: has origin of replication) • everal marker genes • unique cleavage site(s) • for expression, must contain control elements, such as promoters, terminators, ribosome binding sites, etc… Cloning Vectors But 15go in w/out a problem Hybrid ofplasmid and phage chracteristics Two telomeres then centromere ofyeast BAC/YAC – Bacterial/Yeast artificialchromosome BAC more stable thanYAC –easier to work with (yeast has verygood repair mechanisms, willrecognize foreignDNA) Cangrow onmedia w/ amp and tetracycline - ifDNAinsert was inSal1 site –it would destroy the gene conferringtetracyline resistance - select cells that grow onplates containing Ampicillin but die onplates containingtetracycline A plasmid vector, pUC18 Ifput insert into lac z(your insert) goingto interrupe beta- galactosidase whichwillno longer Polylinkerw/ restriction enzymes convert Xgalinto a blue dye Disrupting beta galactosidase =white -subtrate forb- colonies galactosidase - makes a blue No polylinker color =blue colony :disrupts lac Z and willsee white colonies → for genomic DNA, large fragments ofDNA Cloning in phage λ two arms plus insert can only take in 45kb BAMHIremoves pieces ofDNA BAMHIhas two pathways -lytic pathway:replicate and create newprogeny, burst out and
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