Sanger Flashcards
Purpose of DNA sequencing (4)
finding mutation, microorgannisms, human haplotypes, and polymorphisms
whats the difference between sanger ddNTP and deoxynucleotides (dNTP)
2’3’ dideoxynucleotide triphosphates have a hydrogen atom to the 3’ than OH group
what is the purpose of sanger using ddNTP instead of dNTPs
ddNTPs cannot form phosphodiester bond with next dNTP = stops elongation / DNA synthesis
What is used for a Sanger sequencing PCR reaction (5)
Template DNA, DNA pol, primers, all 4 fluorescent labelled ddNTPs, and dNTPs
what are the steps in sanger PCR and what do they do
denaturation = separate DNA
hybridization = attach primers
elongation = bidirectional addition by DNA pol 5’ to 3’
flurescent labelled DNA = ddNTP stops elongation of dNTPs
what step cleans up extra nucelotide sand primers
capillary electrophoresis
what method does capillary electrophoresis use
electrokinetic injection and moves DNA by size to cathode to anode
what are the capilaires filled with and what does it do
polymer - decrease electroosmosis
what is demonstrated in poor PCR quality
bottom has background waves
what causes a spike and then flat lined signal
unincorporated dye
what causes unincorporated dye
insufficient template
thermal cycler malfunction
loss of product during clean up
reagent not added or deteriorated
what causes low signal in electropherograms
failed injection
not enough sample
old buffer
broken capillary
what does a low signal on electrophogram look like
lots of bumps (high frequency)
what causes large peaks in the first 120 bases
poor clean up of reaction = cannot rerun
what causes shoulders (small bump on right of peak) on all peaks
capillary array needs to be replaced
overloaded sample
homopolymeric region in sample
