L19 - Nucleic acid sequencing 1 Flashcards
(11 cards)
what is nucleic acid sequencing
determine/working out the sequence of a target
= deterine all bases in a DNA sequence
describe how sanger sequencing works to identify a sequence
separate strands with heat and add specific primer
= the primer will be the same for all the strand so there is a COMMON 5’ end/start
polymerase copies sequence onto template with dNTPs until a fluorescently labelled terminator ddNTP is added
= does not contain 3’ OH so no more synthesis can occur
product of cycle sequencing is a mix of fluorescently labelled products with COMMON 5’ end and differing 3’ ends/lengths
= sequence can be worked out with gel electrophoresis and detector
how does gel electrophoresis work with sanger sequencing to determine the sequence from a mix of fluorescently labelled fragments
mix is loaded into a single lane of acrylamide gel and separated by electrophoresis
detector at bottom of gel monitors the order of colour coming out bottom
= each ddNTP is a specific colour
= we can then sequence from the order of colours
how can we insert a gene into a plasmid if we don’t know the sequence of the gen
restriction enzymes cut at specific sites
= create sticky ends that glue the vector and insert together
we don’t need to know the sequence of the gene to do this
how do we design primers for sanger sequencing for example if we don’t KNOW the sequence of the gene
we do know the sequence of the plasmid/vector around where the gene was inserted
= So we can design primers that bind to the known plasmid sequence flanking the inserted gene and READ in from known areas
2 limits of read/sequence lengths available to sanger sequencing
ddNTP:dNTP ratio:
we dont want the ddNTP to be put in every time it can otherwise we would never be able to do long reads = same with not being pulled in enough
band diffusion in long electrophoresis:
the longer DNA stays in ge the less ‘tight’ the bands are = DNA diffuses in gel
= overlap of peaks
what is the length of sequencing that sanger can accurately do
500-1000bps
describe which methods of seuqnencing you puld use for a short DNA sequence and a large sequence
short = sanger
large = shotgun
sanger is accurate and precise like a rifle - shotgun fires in every dirction
describe what shotgun sequencing is and how it works
randomly cut DNA into overlapping fragments via sonication and sequence them via sanger
= use overlaps to assemble the full sequence
describe the workflow of sotgun seuqncing
- randomly cut up DNA into overalpping fragments
- run on gel to spereate by size
- insert different sized fragment into plasmids and transform into bacteria
= clone library with each colony having a different sized fragment
- sequence the fragment using ‘universal’ primer as all the vectors are the same only difference is the fragment WITHIN the vector
- computer identifies overlapping regions and works out sequence
why can sanger sequencing only be used to sequence a single DNA region at a time
you need to have a common starting point/5’ end
= different starting points would show a complete jumble with mixed peaks/fluorescence at every fragment size
