Unformatted text preview: Lecture 12 review: DNA replica4on – Experiments Purpose Ques4on Method Experiment 1 What does DNA replica7on in E.coli look like? (1) Add radioac4ve nucleo4des (3H‐ thymidine) to E.coli culture; bacterial cells will uptake and incorporate into replicated DNA (2) Collect cells, lyse to release DNA (3) Visualize DNA via autoradiography (exposing to ﬁlm) LS3‐2 (week 9) Experiment 2 Bi or uni‐direc7onal mode of replica7on? (Eukaryo7c, prokaryo7c genomes) Pulse radiolabeling (1) Give cells pulse of LOW‐radioac7ve precursor (dNTPs) Experiment 3 Uni‐direc7onal mode of replica7on in PLASMID? (1) Use restric4on enzyme to linearize plasmid DNA (2) Observe replica4on via electron microscopy (“home video”) (2) Give cells pulse of HIGH‐radioac7ve precursor (3) Visualize via autoradiography; Only the “most newest” replicated DNA has incorpora4on of HIGH‐radioac4ve NTs. Results/ conclusion “Theta” mode of DNA replica4on in E.coli Distance from origin expands in 1 direc4on = unidirec4onal BI‐direc4onal mode of replica4on Lecture 15: PCR + sequencing LS3‐2, week 9 DNA ampliﬁca4on PCR “regular” PCR RT‐PCR (reverse transcriptase) Make DNA from RNA template • Expression analysis (only at the end of last cycle) • Make cDNA libraries qRT‐PCR (quan4ta4ve real‐4me PCR) Measure ampliﬁca4on of product aber each ampliﬁca4on cycle • Expression analysis; Quan4ta4ve measurement of a sequence of interest in real‐4me Big idea Applica4on Amplify region of DNA Liale lots • Cloning • Sequencing DNA sequencing Sanger method • radioac4vely labeled dNTPs • Run A,G,C,T in diﬀerent lanes v Automated • ﬂuorescently labeled dNTPs (each dNTP w/ diﬀerent color) • Combine and run all reac4ons in one lane; ﬂuorescent signal detected by computer “automa4cally” Lec13 ‐ VECTORS Naturally occurring in… Modiﬁca4ons/requirements for use as a vector Bacteria 1. 2. 3. plasmid λ phage Bacteriophage (virus that infects bacteria) Removal of 1. 2. DNA fragment inserted where? Organism used for replica4on? How do you get recombinant DNA into bacteria? Selec4on? Each arising ____ contains same clone/origin Eﬃciency Amount of foreign DNA it can hold? Applica4on preference Lec13 ‐ VECTORS Naturally occurring in… Modiﬁca4ons/requirements for use as a vector Bacteria plasmid λ phage Bacteriophage (virus that infects bacteria) Removal of 1. Genes required for lysogenic growth 2. Genes non‐essen4al for viral replica4on (i.e. structural) Between leb and right arms bacteria Infec4on 1. ORI (origin of replica4on) 2. Drug resistance gene 3. Polylinker region (lots of restric4on sites) DNA fragment inserted where? Organism used for replica4on? How do you get recombinant DNA into bacteria? Selec4on? Polylinker region – ar4ﬁcal DNA sequence that contains mul4ple restric4on sites Bacteria Transforma4on (2 kinds) • electropora4on • CaPO4 (chemically inducing holes) Drug resistance (3 kinds) aka selec4on markers aka an4bio4cs 1. Ampicillin (amp) = blocks cell wall synthesis) 2. Tetracyclin (tet) = binds 30s ribosomal subunit; interferes with tRNA binding to mRNA = blocks protein synthesis 3. Chloramphenicol (cam) = binds 70s ribosomal subunit = blocks protein synthesis Forma4on of plaques (killing zones) Each arising ____ contains same clone/origin Eﬃciency Amount of foreign DNA it can hold? Applica4on preference Colony Plaque Less Not much; ~3kB Cloning/protein expression More (1000x) A lot; Up to 25kB Genomic libraries Can be done in vitro ...
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- Spring '08
- DNA, Chloramphenicol, 3kb, 25kB