Lecture 10 - DNA Sequencing Flashcards
What does DNA sequencing do
generate the order nucleotides read in a 5’ to 3’ direction
Why do we use DNA sequencing
detecting mutations, typing microorganisms, identifying haplotypes, designating polymorphisms
What is sanger sequencing
using ddNTPs in PCR
What is a ddNTP
deoxynucleotides that have a hydrogen atom attached to the 3’ carbon rather than an OH group
What do DDNTPs do
terminate DNA chain elongation because they cannot form a phosphodiester bond
How are the DDNTPs labeled
each is labelled with a different coloured fluorescent dye
How is sangar sequencing usually run
in a single capillary
What is the workflow of sanger sequencing
- extraction of DNA
- amplification of DNA fragment
- sample preparation
- sanger sequencing reaction
- clean up of sequencing reaction
- capillary electrophoresis
- data analysis
How does injection occur for sanger sequencing
electrokinetic injection
What does the polymer in capillary electrophoresis do
minimize electro-osmosis
What is an electropherogram
raw data peaks are generated into the final sequence of the DNA template as well as the electropherogram with each colored peak representing each letter of the sequence
What are qualities of a good quality sequence
evenly spaced and relatively even peaks
What does low signal indicate
insufficient sample volume
failed injection
old buffer
broken or blocked capillary
What does no signal indicate
insufficient template
thermal cycler malfunction
loss of product during clean up
reagents not added or deteriorated
What do large peaks in the first 120 bases represent
poor clean up of sequencing reaction
