DNA Sequencing

Question 1

Explain dideoxynucleotides

Answer 1

Differ by absence of 3’ hydroxyl from dNTPs
No phosphodiester bound
CHAIN TERMINATORS

Question 2

Explain classical Sanger Sequencing

Answer 2

Radioactively-labelled primer is added to template DNA
4 separate reactions are set up, each with ONE different terminator

Question 3

What is T7 DNA polymerase used for?

Answer 3

Synthesising new strand
- from bacteriophage
- likes both ddNTPS and dNTPS

Question 4

What is found across 4 tubes?

Answer 4

Different terminators where we will get products corresponding to termination at every base in sequence

Question 5

What does each reaction mix contain?

Answer 5

A range of products, according to when terminator was incorporated

Question 6

What denatures ssDNA?

Answer 6

Heat

Question 7

How are products separated?

Answer 7

Separated by size by PAGE

Question 8

How are radioactive products visualised?

Answer 8

By exposure of X-ray film to gel

Question 9

What are the steps in Sanger sequencing?

Answer 9

1. Bind primer to template DNA
2. Set up 4x reaction mixes with different chain terminator
3. Separate fragments by size (PAGE)
4. Visualise products by autoradiography
5. Read DNA sequence from smallest fragment to largest

Question 10

Explain automated DNA sequencing

Answer 10

Use ddNTPS, but each has different fluorophore
Products are separated by PAGE
Laser excited fragments and detector picks up fluorescence

Question 11

Explain capillary system

Answer 11

Capillaries as the separation matrix
Laser beam excited fragments and camera detects fluorescence
Produces electropherogram

Question 12

Explain electropherogram

Answer 12

Displays peaks of particular ddNTPs

Question 13

What is workflow for sequencing?

Answer 13

PCR -> clean up of PCR products -> Sequencing -> Purification -> Capillary electrophoresis

Question 14

What is pyrosequencing?

Answer 14

Form of sequencing that determines genetic sequence based upon measuring which nucleotide is added into growing DNA strand

Question 15

Explain pyrosequencing process

Answer 15

Pol. extends synthesised strand
1 of 4 nucleotides injected
Nucleotide incorporated, PPi groups released
Light detected by camera

Question 16

What does PPi groups produce?

Answer 16

PPi used by sulfurylase = ATP
ATP is used by luciferase to catalyse reaction for light

Question 17

What happens when a nucleotide is incorporated in pyrosequencing?

Answer 17

Chain reaction of enzyme-catalysed reactions that lead to light being produced (light quantity = amount of nucleotides)

Question 18

Explain PCR-based amplification of region of interest and Incubation with streptavidin-sepharose beads

Answer 18

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

Question 19

Explain Immobilisation on filter membrane and Amplicon made single-stranded

Answer 19

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

Question 20

Explain annealing of sequencing primer and Pyrosequencing analysis

Answer 20

Bead/PCR product complexes are shaken off into 96-well plate containing assay specific sequencing primer
Sequencing primer anneals
Plate is pyrosequenced

Question 21

How are pyrosequencing results interpreted?

Answer 21

Heigh of peak corresponds to number of nucleotides in sequence
If 2x as high as normal, two nucleotides incorporated
If 3x, three nucleotides incorporated

Question 22

What is pyrosequencing protocol?

Answer 22

1. Regions of interest must be amplified by PCR
2. PCR products should be purified and prepared for the sequencing reaction

Question 23

What is Next Generation Sequencing?

Answer 23

Sequencing of spatially separated DNA fragments performed massively