Lecture 4

Question 1

Sequencing by synthesis is

Answer 1

the most effective way to synthesis DNA

Question 2

Sanger sequencing?

Answer 2

DNA clones are generated by a standard PCR reaction

Question 3

Polymerase-mediated synthesis is critical to ensure

Answer 3

random terminations, resulting in Varying DNA fragments

Question 4

dNTP vs ddNTP

Answer 4

deoxynucelotidetriosephosphate vs dideoxynucelotidetriophophate

Question 5

What do ddNTP’s cause?

Answer 5

Termination

Question 6

ddNTP are labelled?

Answer 6

Flourescently

Question 7

Gel used in sanger sequencing

Answer 7

Polyacrylamide gel

Question 8

How can we detect the sequence from a gel?

Answer 8

Fragment which travels the least= largest= first nucleotide which correlates to the colour.

Question 9

Mechanical detection of colours of ddNTP’s

Answer 9

Laser beamer, photomultiplier to form an electro gram with the colours present.
Peak=termination

Question 10

Illumina sequencing platform

Answer 10

Next generation

Question 11

Illumina relies on

Answer 11

Dye terminator chemistry

Question 12

BRIDGE AMPLIFICON?

Answer 12

found on glass slide and allows for clonal clusters to accumulate

Question 13

Method of illumina sequencing.

Answer 13

1- DNA attaches to a flow cell
(a flow cell has multiple primers)
2- DNA forms a bridge with another primer
3- PCR HEAT UP, will continue to replicate and form a cluster

Question 14

Reversible dye terminator chemistry: FUNCTION OF REVERSIBLE TERMINATOR

Answer 14

blocks the addition of another NT.

Question 15

F group in reversible dye terminator chemistry

Answer 15

Chorophore group which is cleavable if the correct chemical Is added

Question 16

When F cleaved

Answer 16

Light given off which can be detected by a camera

Question 17

Advantage of Illumina sequencing

Answer 17

Multiple clusters are doing the same cloning at the same time therefore a quick process.
- Few hours

Question 18

SMRT stands for

Answer 18

single molecule Real time sequencing

Question 19

what kind of technique is SMRT?

Answer 19

Next generation

Question 20

What does SMRT allow direct detection of?

Answer 20

DNA modifications.

Question 21

Method of SMRT sequencing

Answer 21

1- Plate with wells in
2- DNA polymerase is attached to the bottom of the well
3- Zero-mode waveguide: which illuminates the proportion in which DNA polymerase is sat in
4- If the correct nucleotide approaches the DNA polymerase, it is held in the active site long enough for. flash of light to be given off, which is detected by a camera

Question 22

Chemistry behind SMRT sequencing

Answer 22

Addition of a chlorophore group on dNTP (at end of tri phosphate) when diphosphate is cleaved light is given off

Question 23

Delay in light given off indicates?

Answer 23

Hindered rate in which that nucleotide is passing through DNA polymerase, after the delay, a modification occurs

Question 24

most widely used NGS platform?

Answer 24

ILLUMINA