2. Emerging Technologies

Question 1

‘ingredients’ in Sanger sequencing

Answer 1

-DNA template
-primers
-DNA polymerase
-all 4 dNTPs (in each tube)
-1 type of ddNTP (1/100th concentration of dNTPs)

Question 2

steps in Sanger/dideoxy/dye terminator sequencing

Answer 2

1. template denatured (ss)
2. 3’ primer anneals to template
3. 4 separate reactions set up (each with a different ddNTP)
4. DNA synthesis terminated whenever a ddNTP is incorporated
5. PCR produces DNA fragments of varying lengths, each ending with a ddNTP
6. electrophoresis
7. fluorescent imaging

Question 3

‘ingredients’ of pyrosequencing

Answer 3

-DNA template
-bead/substrate
-primer
-DNA polymerase
-ATP sulfurylase
-luciferase
-apyrase
-adenosine 5’ phosphosulfate
-luciferin

Question 4

explain fluorescing in pyrosequencing

Answer 4

-as a nucleotide is added to the chain, 2 of it’s phosphates are cleaved off
-2 phosphates = pyrophosphate
-pyrophosphate converted to ATP
-ATP used by luciferin to produce light

Question 5

why is pyrosequencing also known as ‘sequencing by synthesis’

Answer 5

because every time a nucleotide is added to the growing strand, light is emitted which can be detected

each nucleotide has a characteristic amount of light produced -> sequencing

Question 6

workflow of conventional sequencing

Answer 6

1. fragment DNA
2. ligate products to plasmids
3. amplify inside bacteria
4. analysis

Question 7

workflow of second generation sequencing

Answer 7

1. fragment DNA
2. ligate to short oligos
3. literally roll ligated DNA-oligos onto glass slide
4. allow binding wherever
5. sequencing

-can all be performed in the same tube unlike conventional sequencing

Question 8

steps of NGS

Answer 8

1. fragment DNA and ligate universal adaptors
2. amplify single molecules (beads vs free)
3. sequence clonal amplicons
4. computer assemble data