mol6 - BIMM 100 Ch. 5 (cont d) Using Molecular Biology:...

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Unformatted text preview: BIMM 100 Ch. 5 (cont d) Using Molecular Biology: Recombinant DNA fnish chapter by midterm Remember: Classic Experiments and Animations on www.whFreeman.com/lodish6E vectors (plasmids) restriction endonucleases, DNA ligases poly-linkers libraries (oligos) cloning fnding a clone/gene defning (describing/sequencing) a clone PCR learning From hybridization: blots, in situs , arrays ....... expression cloning Libraries in recombinant DNA studies collections of clones genomic vs. cDNA insert size considerations/ vector limits representation ( copies of mature mRNA as DNA ) (uses polyA tails) [20 kb plasmid - phage - cosmid - P1 phage - BAC -YAC 2 Mb] How many clones to represent a genome? How to Fnd THE one of interest? (for section/OH) chemical synthesis of oligonucleotides important for cloning, sequencing, and PCR and ....... chemical, non-enzymatic synthesis synthesis is opposite polarity of in vivo synthesis! Old MCB 9-18 ( oligos ) (3 5 synthesis) A few words ~ Oligonucleotides Building a cDNA library in a plasmid vector Enzyme: Reverse transcriptase MCB 5-15 Building a cDNA library in a vector - 2 Digest with Eco RI Building a cDNA library in a vector - 3 fnding a clone oF interest The principle: complementary sequences will hybridize Output: read on X-ray flm MCB 5-16 The assay: membrane-based hybridization Using the library: by sequence Probe MCB 5-16 fnding a clone oF interest the practice: screening a library plasmid vector-based library in E. coli host cells replicate colonies onto membrane lyse cells -- DNA sticks, is denatured, retained in place hybridize with * probe (next), wash, detect, pick * What about * probes? key for identiFcation and cloning previously identiFed -- comparative cloning (cross species, gene families) synthesized -- based on known/inferred properties degenerate probes -- based on protein sequence unique probes -- based on known sequences expression cloning: antibodies or activities genetic approach -- mutant phenotype (complementation) What about...
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mol6 - BIMM 100 Ch. 5 (cont d) Using Molecular Biology:...

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