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Lecture 11

BIOL 5060 Lecture 11: 2:21_recombinantDNATech_hoffman

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BIOL 5060

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2/21_RecombinantDNATech_Hoffman 2/21/2017 3:04:00 PM Site directed mutagenesis • Requires a narrow specific hypothesis, you’re going to direct a specific mutation to your clone and see what the consequence is • Procedure o Oligonucelotides are primers that contain the mutation of interest ▪ Use them to resynthesize your clone new clone will have the mutation ▪ Gene to be cloned is expected to be important for regulation or activity  Mutation may up-regulate/ down-regulate activity or maybe even inactivate it  We are concerned with regulatory sites especially phosphorylation sites= most common regulatory site to rapidly change the function of a protein  Ser and Thr= most commonly phosphorylated sites   Changed to 1) alanine non phosphorylat-able version of the protein • 2) aspartic acid or glutamic acid phosmomimetic (mimics phosphorylated form) ▪ 1) Find your codon of interest and make 2 oligonucleotides of interest***  REMEMBER THIS: 2 olligonucleotides represent the two strands but with the alteration in the center of the oligos  Altered codon is in the middle; roughly 14-17 nt to either side  Design oligos and use them to replicate plasmid in vitro (like PCR in that it involves heating up the DNA to melt it apart, cooling it to allow it to anneal to the new oligos and synthesizing the new strand)  There is no ligase so, there is a niche (its not a circle)  Altered codon is relatively small compared to flanking sequences (oligos) • We will do a couple rounds of this to make a few strands o Different from PCR in that the new strand will never be used for the synthesis of a new strand… o WHEN the product becomes the substrate= PCR o Here, the product never become the substrate o The substrate is always the original plasmid o The product is the complementary strand with the mutation you designed • This allows for… o Mismatched oligote to match with its almost complement ▪  it will anneal but not circularize o We will end up with more product strands than template strands o 30 bp will overlap and anneal—E. Coli will see the nicks in the circular DNA and synthesize enzymes to seal them to make completely double stranded plasmid • After, we are left with parental plasmid and complementary with mutation o TRICK to enrich for mutant plasmids: before transformation, we will digest the DNA with DpnI o DpnI is SPECIAL b/c it cuts GATC= a= dam methylated site ▪ It cuts dam methylated sites of hemi-methylated sites ▪ Doesn’t cut un-methylated ▪ Selectively digest wild-type/ parental plasmid DNA  Will not digest the mutated ones  = a way to make sure we are left with only mutated DNA; largely shifts us toward the mutants of interest (without dam methylated site) o Another TRICK to finding plasmids with mutation ▪ Example: in situations where given a wobble nucleotide, change it to create a restriction enzyme site… rd  Wobble base= 3 NT  Restriction enzyme site= preferably a 6 base cutter (more rare) ▪ Sequence candidate plasmid  Confirm mutation of interest  No other mutations ▪ You’re putting your plasmid of interest into DpnI digest and then using this strand as a parental strain to sequence a mutated gene o Methylation doesn’t always block restriction digestion (although most often it does) – doesn’t always have to block digestion ▪ It will digest all the original plasmids- but there was no dam methylase in the synthesis reaction only in the E.coli cell ▪ It will not affect the plasmid in the in vitro mutagenesis reaction that carries the mutation that you designed into the oligos way to find only plasmids with the mutation PCR****- more in depth; more in use now-a-days; = cornerstone of molecular biology that we need to understand it in order to make things work well • Need to know how to design good PCR Oligos • Polymerase Chain Reaction o A type of in vitro DNA replication amplification o Will differ in the way we design the Oligos leads to exponential amplification (Magic in PCR) because the product becomes the template ▪ Product becomes the substrate as the reaction goes
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