Sequencing Flashcards
Gibson assembly and benefits
Assembly of multiple linear DNA fragments. Multiple overlapping DNA fragments can be joined in a single isothermal reaction regardless of fragment length/ end compatibility.
Product of gibson assembly
Ligated double-stranded DNA molecule
How many enzymes gibson assembly?
3
General mechanism of Gibson assembly:
- Form 3’ single stranded overhangs with T5 exonuclease -> unstable at 50C + exposes 3’end after time
- Anneal complimentary termini 50 C with Taq ligase + phusion polymerase
- Repair at 50 C with phusion polymerase (high fidelity) + taq ligase -> fill gaps
- Nick-sealing with Taq DNA ligase
When do we use Gibson assembly?
Simultaneous assembly of up to 8-10 fragments
- can be short fragments (annealed oligos without PCR amplification)
- Vector can be PCR-amplified. linearised by rest. enzymes
What is the first protein to be sequenced?
Insulin 1949-50- linear polypeptides
Early challenges with sequencing
- Different DNA molecules chemically similar so difficult to separate
- Chain length DNA > protein - complete sequence unapproachable
- aa varying properties
- No base-specific DNAses known
When was the first DNA sequences?
1968 finished 1971 lambda cos ends
mechanism of early sequencing:
- Repair reaction from 3’OH end (E.coli polymerase) using radioactive nucleotide
- Nuclease degradation
- Isolation of synthesised oligonucleotide
- Sequence determination
Advantages/ disadvantages of Sanger 1975
Adv: rapid + allowed seq. phix174
Dis: ssDNA + accuracy -8 reaction -> confirmatory data
Advantages/ disadvantages of Gilbert 1977
Adv: DsDNA - 4 reactions sufficient
Dis: Strand separation
Advantages/ disadvantages of chain termination 1977
Adv: Increase accuracy - only 4 reactions
Dis: ssDNA - phage M13 - alkaline denaturation
Dideoxyribonucleoside triphosphate
similar to deoxy but prevents strand formation at 3’ end by removing 3’OH
Materials required for chain termination
SS DNA template - to be sequenced
- oligonucleotide primer complementary to upstream region of template DNA P
Which enzyme is used for chain termination and why
Klenow fragment -> lacks 5’ 3’ exonuclease activity
What happens when too much or too little ddNTP is put in the mixture?
Short products / long products
At end of chain termination reaction…
formide added for denaturation and strands heated to separate from template
-> loaded onto gel + separated by gel electrophoresis
Why is urea used in chain termination?
Minimise DNA secondar structure -> prevent affect on electrophoretic mobility
How are chain termination sequences read?
On large sheets of X-ray produced from autoradiography of DNA bands -> seq read from bottom
beginning 100 nt/ lane -> 350 using shark tomb comb + 32P
How was chain termination automated? What was no longer needed?
Dye-terminator sequencing -> only 1 reaction to take place so primer isn’t needed since ddNTP labelled with fluorescent tag
Where does automated chain termination take place?
Performed in PCR machine
What replaced the gel in automated chain termination? and why?
Capillary electrophoresis. Separation base on total charge (5-20 kv), polymer solution replaces need of manually paired gel, elecktrokinetic injection for sample loading -> 1000nt/ capillary/ nn read
Problems with Sanger:
- Gels/ polymers as separate media
- Limited # of sequence handled in parallel
- Difficulties in complete automation of sample prep.
Next-gen sequencing
Parallel handling of samples -> shorter reads with less accuracy for single read
=> higher degree of sequence coverage so final = highly accurate
- years -> weeks in sequence true 1k in days (2014)
Next gen sequencing mech/ pyrosequencing:
- dNTP is attached to 3’ end of growing DNA, 2 phosphates are released as pyrophosphate (ppi)
- Ppi + adenosine 5’ phosphate -> ATP using ATP sulfurylase
- ATP used to convert lucifernin to oxyluciferin by luciferase
- Produces light proportinal to amount of ATP + thereby proportional to amount of NT added to DNA
- Light detected by a CCD
In the solid phase of next gen has how many enzymes?
3
Liquid phase of next gen consists of :
3 enzymes and apyrase
Apyrase
Hydrolyses ATP + unincorporated dNTP which switches off light production
2 Apyrase reactions
dNTP -: dNDP + dNMP + phosphate
ATP -> ADP + AMP + phosphate
In the pyrosequencing reaction set up:
4 dNTP’s added one at a time with apyrase degradation/ washing in between.
- > Amount of light proportional to # nucleotide added
- 454 sequencer cycles between 4dNTPs to build sequence
- > 300-700 nt of seq. can be read which is short + less accurate than Sanger
How is the library for 454 genome sequencer?
- DNA sheared (300-800 bp) ends are ‘blunted’
- 2 diff adaptors added to each end of fragment
- 1 is complimentary to oligonucleotide on seq. bead
- > beads: DNA controlled
- > most beads only get a single DNA
- > oil added to beads -> emulsion
Benefits of 454 genome sequencer
Each bead coated with 1 mill identical copies of original DNA
- mill’s of clonally amplified seq. templates on each bead + no cloning + colony picking ( fast + no bias towards specific clones)
Pros and cons of 454 sequencer
Pro: no cloning, low average sub error rate
Cons: average sub error rate higher than Sanger, higher backing error b/c i.vitro amps preferred for seq.
- bead prep: fraction carry copies of multiple diff seq.
-> loss of synchrony -> echo
Illumina sequencing
- Prep. genomic DNA:
- randomly fragment genomic DNA + ligate 2 different adapters to both ends of fragments - Attach DNA to surface
- > surface flow cell has 2 population’s of immobilised oligont’s complimentary to adaptor ends - Bridge amplification
- > unlabelled NT + enzyme -: initiate solid-phase bridge amplification x 35 - Denature DS molecule
Data acquisition for illumina
1st base determined by:
Adding all 4 labelled irreversible terminants, primers + DNAP to flow cell for 1st seq cycle
-> laser excitation
-> image of emitted fluorescence from each cluster
-> base recorded
-> blocked 3’ terminus + fluorophore from each included base removed
Pros and Cons of illumina
Pros: no cloning, lib+ flow cell prep includes in vitro amplification steps, error: 10-2 - 10-3, 26 nt -> 100nt
cons: later positions less accurate due to bi-directional phasing
- sim. detection of 4 different fluorescent dyes with sim. emission spectra contributes to error
- despite higher error rate + considerable shorter read length 5k mb. day for 0.5$/Mb obtained
Nanopore
Electrically resistant polymer membrane which ionic current is passed through
