DNA Sequencing Flashcards
Explain dideoxynucleotides
Differ by absence of 3’ hydroxyl from dNTPs
No phosphodiester bound
CHAIN TERMINATORS
Explain classical Sanger Sequencing
Radioactively-labelled primer is added to template DNA
4 separate reactions are set up, each with ONE different terminator
What is T7 DNA polymerase used for?
Synthesising new strand
- from bacteriophage
- likes both ddNTPS and dNTPS
What is found across 4 tubes?
Different terminators where we will get products corresponding to termination at every base in sequence
What does each reaction mix contain?
A range of products, according to when terminator was incorporated
What denatures ssDNA?
Heat
How are products separated?
Separated by size by PAGE
How are radioactive products visualised?
By exposure of X-ray film to gel
What are the steps in Sanger sequencing?
- Bind primer to template DNA
- Set up 4x reaction mixes with different chain terminator
- Separate fragments by size (PAGE)
- Visualise products by autoradiography
- Read DNA sequence from smallest fragment to largest
Explain automated DNA sequencing
Use ddNTPS, but each has different fluorophore
Products are separated by PAGE
Laser excited fragments and detector picks up fluorescence
Explain capillary system
Capillaries as the separation matrix
Laser beam excited fragments and camera detects fluorescence
Produces electropherogram
Explain electropherogram
Displays peaks of particular ddNTPs
What is workflow for sequencing?
PCR -> clean up of PCR products -> Sequencing -> Purification -> Capillary electrophoresis
What is pyrosequencing?
Form of sequencing that determines genetic sequence based upon measuring which nucleotide is added into growing DNA strand
Explain pyrosequencing process
Pol. extends synthesised strand
1 of 4 nucleotides injected
Nucleotide incorporated, PPi groups released
Light detected by camera
What does PPi groups produce?
PPi used by sulfurylase = ATP
ATP is used by luciferase to catalyse reaction for light
What happens when a nucleotide is incorporated in pyrosequencing?
Chain reaction of enzyme-catalysed reactions that lead to light being produced (light quantity = amount of nucleotides)
Explain PCR-based amplification of region of interest and Incubation with streptavidin-sepharose beads
Short region of DNA is amplified by PCR
PCR products are incubated with streptavidin-sepharose beads
1/2 primers has 5’ biotin label
Biotin from primer sticks the PCR product to beads
Explain Immobilisation on filter membrane and Amplicon made single-stranded
A vacuum is applied through a filter
PCR product/bead complexes are immobilised
DNA is denatured by immersion in sodium hydroxide to become single stranded
Only strand with biotinylate primer remains
Explain annealing of sequencing primer and Pyrosequencing analysis
Bead/PCR product complexes are shaken off into 96-well plate containing assay specific sequencing primer
Sequencing primer anneals
Plate is pyrosequenced
How are pyrosequencing results interpreted?
Heigh of peak corresponds to number of nucleotides in sequence
If 2x as high as normal, two nucleotides incorporated
If 3x, three nucleotides incorporated
What is pyrosequencing protocol?
- Regions of interest must be amplified by PCR
- PCR products should be purified and prepared for the sequencing reaction
What is Next Generation Sequencing?
Sequencing of spatially separated DNA fragments performed massively
