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Neur2001 > Sequencing > Flashcards

Sequencing Flashcards

1
Q

Gibson assembly and benefits

A

Assembly of multiple linear DNA fragments. Multiple overlapping DNA fragments can be joined in a single isothermal reaction regardless of fragment length/ end compatibility.

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2
Q

Product of gibson assembly

A

Ligated double-stranded DNA molecule

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3
Q

How many enzymes gibson assembly?

A

3

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4
Q

General mechanism of Gibson assembly:

A
  1. Form 3’ single stranded overhangs with T5 exonuclease -> unstable at 50C + exposes 3’end after time
  2. Anneal complimentary termini 50 C with Taq ligase + phusion polymerase
  3. Repair at 50 C with phusion polymerase (high fidelity) + taq ligase -> fill gaps
  4. Nick-sealing with Taq DNA ligase
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5
Q

When do we use Gibson assembly?

A

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
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6
Q

What is the first protein to be sequenced?

A

Insulin 1949-50- linear polypeptides

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7
Q

Early challenges with sequencing

A
  1. Different DNA molecules chemically similar so difficult to separate
  2. Chain length DNA > protein - complete sequence unapproachable
  3. aa varying properties
  4. No base-specific DNAses known
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8
Q

When was the first DNA sequences?

A

1968 finished 1971 lambda cos ends

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9
Q

mechanism of early sequencing:

A
  1. Repair reaction from 3’OH end (E.coli polymerase) using radioactive nucleotide
  2. Nuclease degradation
  3. Isolation of synthesised oligonucleotide
  4. Sequence determination
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10
Q

Advantages/ disadvantages of Sanger 1975

A

Adv: rapid + allowed seq. phix174
Dis: ssDNA + accuracy -8 reaction -> confirmatory data

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11
Q

Advantages/ disadvantages of Gilbert 1977

A

Adv: DsDNA - 4 reactions sufficient
Dis: Strand separation

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12
Q

Advantages/ disadvantages of chain termination 1977

A

Adv: Increase accuracy - only 4 reactions
Dis: ssDNA - phage M13 - alkaline denaturation

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13
Q

Dideoxyribonucleoside triphosphate

A

similar to deoxy but prevents strand formation at 3’ end by removing 3’OH

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14
Q

Materials required for chain termination

A

SS DNA template - to be sequenced

- oligonucleotide primer complementary to upstream region of template DNA P

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15
Q

Which enzyme is used for chain termination and why

A

Klenow fragment -> lacks 5’ 3’ exonuclease activity

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16
Q

What happens when too much or too little ddNTP is put in the mixture?

A

Short products / long products

17
Q

At end of chain termination reaction…

A

formide added for denaturation and strands heated to separate from template
-> loaded onto gel + separated by gel electrophoresis

18
Q

Why is urea used in chain termination?

A

Minimise DNA secondar structure -> prevent affect on electrophoretic mobility

19
Q

How are chain termination sequences read?

A

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

20
Q

How was chain termination automated? What was no longer needed?

A

Dye-terminator sequencing -> only 1 reaction to take place so primer isn’t needed since ddNTP labelled with fluorescent tag

21
Q

Where does automated chain termination take place?

A

Performed in PCR machine

22
Q

What replaced the gel in automated chain termination? and why?

A

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

23
Q

Problems with Sanger:

A
  • Gels/ polymers as separate media
  • Limited # of sequence handled in parallel
  • Difficulties in complete automation of sample prep.
24
Q

Next-gen sequencing

A

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)

25
Q

Next gen sequencing mech/ pyrosequencing:

A
  1. dNTP is attached to 3’ end of growing DNA, 2 phosphates are released as pyrophosphate (ppi)
  2. Ppi + adenosine 5’ phosphate -> ATP using ATP sulfurylase
  3. ATP used to convert lucifernin to oxyluciferin by luciferase
  4. Produces light proportinal to amount of ATP + thereby proportional to amount of NT added to DNA
  5. Light detected by a CCD
26
Q

In the solid phase of next gen has how many enzymes?

A

3

27
Q

Liquid phase of next gen consists of :

A

3 enzymes and apyrase

28
Q

Apyrase

A

Hydrolyses ATP + unincorporated dNTP which switches off light production

29
Q

2 Apyrase reactions

A

dNTP -: dNDP + dNMP + phosphate

ATP -> ADP + AMP + phosphate

30
Q

In the pyrosequencing reaction set up:

A

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
31
Q

How is the library for 454 genome sequencer?

A
  • 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
32
Q

Benefits of 454 genome sequencer

A

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)

33
Q

Pros and cons of 454 sequencer

A

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

34
Q

Illumina sequencing

A
  1. Prep. genomic DNA:
    - randomly fragment genomic DNA + ligate 2 different adapters to both ends of fragments
  2. Attach DNA to surface
    - > surface flow cell has 2 population’s of immobilised oligont’s complimentary to adaptor ends
  3. Bridge amplification
    - > unlabelled NT + enzyme -: initiate solid-phase bridge amplification x 35
  4. Denature DS molecule
35
Q

Data acquisition for illumina

A

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

36
Q

Pros and Cons of illumina

A

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

37
Q

Nanopore

A

Electrically resistant polymer membrane which ionic current is passed through

Neur2001 (10 decks)

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