Pyrosequencing

Question 1

What is the main hindrance of Sanger sequencing?

Answer 1

They require a DNA size determination step

Question 2

What is the issue with Sanger sequencing requiring a size determination step?

Answer 2

It constrains the number of DNA clones that can be sequenced in a single sequencing run

Question 3

What was the early aim of NGS development?

Answer 3

To come up with a method that relied only upon light detection

Question 4

What is the first step of pyrosequencing?

Answer 4

Clonal amplification of DNA fragments

Question 5

What is the first stage of clonal amplification?

Answer 5

Fragmenting the genome into tiny pieces

Question 6

What is ligated onto the ends of the tiny fragments of DNA?

Answer 6

Adaptors

Question 7

What are adaptors in clonal amplification?

Answer 7

Bits of DNA of known sequence that you can design primers against

Question 8

What does mixing water and oil together result in?

Answer 8

Water droplets suspended in oil

Question 9

How many water droplets are there?

Answer 9

Far more than the number of DNA molecules

Question 10

Why are there so many water droplets in clonal amplification?

Answer 10

So only a single DNA fragment will enter each droplet

Question 11

What added to each droplet once the DNA fragment is in there?

Answer 11

PCR equipment–> polymerase, buffers, primers (complementary to adaptors)

Question 12

What does each water bead act as?

Answer 12

A microreactor

Question 13

How does each water droplet act as a microreactor?

Answer 13

One PCR reaction (for each DNA fragment) occurs in each droplet

Question 14

What are the beads of water spread onto once the PCR is completed

Answer 14

A pico-titer plate

Question 15

What is on pico-titer plates?

Answer 15

A million wells per plate, each that can hold one water droplet

Question 16

Where do the water droplets go in the pico-titer plates?

Answer 16

Each droplet goes into one of the wells

Question 17

What is the second step of pyrosequencing?

Answer 17

Pyrosequencing

Question 18

Where is the pyrosequencing done?

Answer 18

In the pico-titer plates

Question 19

First step of pyrosequencing?

Answer 19

One type of nucleotide is added to the wells at a time

Question 20

What happens when the correct nucleotide is added (i.e. one that is complementary)?

Answer 20

Pyrophosphate is released

Question 21

Why is pyrophosphate released when the correct nucleotide is added?

Answer 21

It is a byproduct of incorporating a nucleotide into DNA

Question 22

What is pyrophosphate a substrate for?

Answer 22

Sulfurylase enzyme

Question 23

What does sulfurylase do to pyrophosphate?

Answer 23

Converts it into ATP

Question 24

What is done with the ATP made from pyrophosphate?

Answer 24

Luciferase uses it to generate a flash of light

Question 25

What does a flash of light during pyrosequencing indicate?

Answer 25

A successful addition of a nucleotide

Question 26

What is added after each nucleotide is added in pyrosequencing?

Answer 26

Apysase enzyme

Question 27

Why is apyrase enzyme added after each nucleotide?

Answer 27

To degrade the nucleotide so it doesnt confuse the machine

Question 28

Why may the intensities of light produced by pyrosequencing be different?

Answer 28

Multiple of the same nucleotide may be added consecutively

Question 29

What two bits of information about the flashes of light are required to determine the sequence in pyrosequencing?

Answer 29

The order of flashes, and their intensity