Sequencing

Question 1

Gibson assembly and benefits

Answer 1

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

Question 2

Product of gibson assembly

Answer 2

Ligated double-stranded DNA molecule

Question 3

How many enzymes gibson assembly?

Answer 3

3

Question 4

General mechanism of Gibson assembly:

Answer 4

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

Question 5

When do we use Gibson assembly?

Answer 5

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

Question 6

What is the first protein to be sequenced?

Answer 6

Insulin 1949-50- linear polypeptides

Question 7

Early challenges with sequencing

Answer 7

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

Question 8

When was the first DNA sequences?

Answer 8

1968 finished 1971 lambda cos ends

Question 9

mechanism of early sequencing:

Answer 9

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

Question 10

Advantages/ disadvantages of Sanger 1975

Answer 10

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

Question 11

Advantages/ disadvantages of Gilbert 1977

Answer 11

Adv: DsDNA - 4 reactions sufficient
Dis: Strand separation

Question 12

Advantages/ disadvantages of chain termination 1977

Answer 12

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

Question 13

Dideoxyribonucleoside triphosphate

Answer 13

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

Question 14

Materials required for chain termination

Answer 14

SS DNA template - to be sequenced

- oligonucleotide primer complementary to upstream region of template DNA P

Question 15

Which enzyme is used for chain termination and why

Answer 15

Klenow fragment -> lacks 5’ 3’ exonuclease activity

Question 16

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

Answer 16

Short products / long products

Question 17

At end of chain termination reaction…

Answer 17

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

Question 18

Why is urea used in chain termination?

Answer 18

Minimise DNA secondar structure -> prevent affect on electrophoretic mobility

Question 19

How are chain termination sequences read?

Answer 19

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

Question 20

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

Answer 20

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

Question 21

Where does automated chain termination take place?

Answer 21

Performed in PCR machine

Question 22

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

Answer 22

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

Question 23

Problems with Sanger:

Answer 23

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

Question 24

Next-gen sequencing

Answer 24

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)

Question 25

Next gen sequencing mech/ pyrosequencing:

Answer 25

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

Question 26

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

Answer 26

3

Question 27

Liquid phase of next gen consists of :

Answer 27

3 enzymes and apyrase

Question 28

Apyrase

Answer 28

Hydrolyses ATP + unincorporated dNTP which switches off light production

Question 29

2 Apyrase reactions

Answer 29

dNTP -: dNDP + dNMP + phosphate | ATP -> ADP + AMP + phosphate

Question 30

In the pyrosequencing reaction set up:

Answer 30

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

Question 31

How is the library for 454 genome sequencer?

Answer 31

- 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

Question 32

Benefits of 454 genome sequencer

Answer 32

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)

Question 33

Pros and cons of 454 sequencer

Answer 33

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

Question 34

Illumina sequencing

Answer 34

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

Question 35

Data acquisition for illumina

Answer 35

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

Question 36

Pros and Cons of illumina

Answer 36

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

Question 37

Nanopore

Answer 37

Electrically resistant polymer membrane which ionic current is passed through