Biology 1002B Lecture Notes - Lecture 22: Reporter Gene, Antimicrobial Resistance, Ampicillin
29 Apr 2018
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Plasmid Vector
• Contains antibiotic resistance genes such as ampicillin gene
o Not all cells took up plasmid vector
o Put treated cells in ampicillin, the cells that survive are the ones that took up the
genes because they have the antibiotic resistance
o Selectable marker - only the cells that are transformed are selected for growth
PCR amplifies copy number of a specific sequence
• Make billions copies of a very specific DNA sequence
• Which of the following characteristics of Taq polymerase makes it especially well suited
for use in PCR reactions?
o It does not need a 3'OH in order to extend DNA strands
• False
o It can recognize and amplify only specific gene sequences
• PCR does this already, we do not use Taq polymerase because of this
o It does not need a template like "regular" polymerases
• False
o It remains functional at high temperatures
• TRUE of Taq polymerase
• Taq polymerase must not degrade at high temperatures because PCR relies on thermal
cycling (increasing and lowering of temperature), so we need a stable enzyme
o First step is to heat up to separate double stranded DNA
o Then after we add DNA primers (NOT RNA PRIMER!!) and let Taq polymerase extend
the strands
• We make these DNA primers ourselves with a synthesizer
• DNA more stable than RNA
PCR Mechanism
• First round of PCR is messy - first new molecules are too long because synthesis goes past
gene of interest
• Next cycles we get fragments that are of desired length
• By cycle 3, we see double stranded DNA that is exactly what we want
o In cycle 2, one strand of the double strands are still too long
o In cycle 3, we get two molecules that match the target DNA sequence
o Every cycle after 3, molecules of target DNA sequence increase exponentially
PCR Primer Design
• To do PCR, you need primers to prime and replicate BOTH top and bottom strand
• To synthesize bottom strand primer, need sequence that is the same as start of top strand
• To synthesize top strand primer, need sequence that is COMPLEMENTARY and
ANTIPARALLEL to the end of the top strand
Various Technologies Deliver Transgenes to Nuclei or Organelles
• There are different ways to get any kind of DNA into any kind of cell
Reporter Genes Confirm Uptake
• Add reporter genes to vectors to know if genes are inserted
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
It does not need a 3"oh in order to extend dna strands. It can recognize and amplify only specific gene sequences: pcr does this already, we do not use taq polymerase because of this. It does not need a template like regular polymerases. In cycle 2, one strand of the double strands are still too long. In cycle 3, we get two molecules that match the target dna sequence: every cycle after 3, molecules of target dna sequence increase exponentially. Antiparallel to the end of the top strand. Various technologies deliver transgenes to nuclei or organelles: there are different ways to get any kind of dna into any kind of cell. Reporter genes confirm uptake: add reporter genes to vectors to know if genes are inserted, reporter gene is yfp in neurons, which neurons that took up this gene will fluoresce yellow.
