DNA sequencing

Question 1

In DNA
sequencing
we are trying
to find out …

Answer 1

…the order of bases in a strand.

Question 2

WHat are the uses of dna sequencing?

Answer 2

• To identify genetic mutations, predict disease risk, and prognosis. such as BRCA1 and BRAC2 in breast cancer.
• Infection disease monitoring
  Gene cloning - to check your sequence after isolation and/or aplification.

Question 3

slide 6

Question 4

What are the two types of DNA sequencing methods?

Answer 4

1) Chain-termination sequencing
2) Next-generation sequencing.

Question 5

How do DNA polymers grow?

Answer 5

by addition of nucleotides at the 3’ end ie sugar phosphate backbone.

Question 6

Addition of a nucleotide results in….

Answer 6

…the production of a pyrophosphate molecule (outer two or β and
γ, P-O-P)

Question 7

What does addition of a nucleotide result in the loss of?

Answer 7

loss of the OH group at the 3’
terminus to form a phosphodiester bond (this
incorporates the α phosphate of the incoming
deoxyribonucleotide).

Question 8

In growth of DNA polymers, how doe shydrogen bonding occur?

Answer 8

Occurs between nitrogenous bases (adenine, guanine, thymine, cytosine, holding the strands together.

Question 9

Why does the 3’ end of the growing DNA chain continue growing?

Answer 9

It keeps growing because each nucleotide that’s added has its own free 3’ OH end ( dATP)

Question 10

If you add a nucleotide in which this OH is
missing the chain will …

Answer 10

… terminate at that point.

Question 11

Termination of the chain growth is utilised in…

Answer 11

…sequencing - to stop the process of growing the chain.

Question 12

Nucleotides can be made to achieve termination of chain growth known as…

Answer 12

dideoxynucleotides (ddATP)

Question 13

DNA fragments can be separated by
electrophoresis according to their ….

Answer 13

….size

Question 14

In electrophoresis, DNA can be pulled towards a positive fields as it has a…

Answer 14

…-ve charge (phosphate groups), and moves towards the +ve anode during electrophoresis.

Question 15

in electrophoresis, the larger the fragment, the slower it …

Answer 15

… runs through a gel (DNA ladder).

Question 16

If you combine DNA chain elongation termination (adding nucleotides with no free OH (ddATP) with the above size separation technique you can…

Answer 16

…order the fragments
and then find out the sequence of nucleotides / bases.

Question 17

PCR allows….

Answer 17

…strands to build up, adding a base at a time.

Question 18

If we carry out PCR with
dideoxynucleotides (ddATP)
(no free OH) we can…

Answer 18

… make the PCR terminate.

Question 19

If we ‘spike’ in small amounts of one of the bases
as the dd format (e.g ddATP Adenine - no free OH)
When ddATP Adenine base pairs the PCR …

Answer 19

… terminates, leaving ‘A’ at the end of the sequence.

Question 20

By running PCR reactions with each of the 4 ddA, ddT, ddG, ddC (dideoxynucleotides), we will force the PCR to…

Answer 20

…terminate when each of the bases are inserted..

Question 21

Labelling dideoxynucleotides (ddATP), using for example colour / floursecence means that we can then determine …

Answer 21

…. which bae the strand of the amplified DNA ended with.

Question 22

Sorting by size then reveals…

Answer 22

… the sequence order.

Question 23

What is capillary electrophoresis?

Answer 23

To sort by size the fragments in the four sequencing reactions are
electrophoresed in denaturing SDS polyacrylamide gel for size separation

Question 24

slide 12

Question 25

The bands detected through capillary electrophoresis are transformed into an ...

Answer 25

...electropherogram.

Question 26

What does each peak relate to in an electropherogram?

Answer 26

each peak relates to the particular fluorescent signal that was detected at a certain time during the capillary electrophoresis run

Question 27

Slide 14

Question 28

When was the first complete genome published?

Answer 28

2022

Question 29

To clone a gene and express the protein it encodes, what needs to be checked?

Answer 29

To clone a gene and express the protein it encodes – vital to check that the sequence is correct otherwise the protein will be wrong

Question 30

Slide 17

Question 31

Define NGS.

Answer 31

term used to describe a number of different modern sequencing technologies

Question 32

What can NGS be used to do?

Answer 32

entire human genome can be sequenced within a single day

Question 33

slide 20

Question 34

Example of NGS Technology ?

Answer 34

illumina Sequencing

Question 35

slide 22

Question 36

What is illumina sequencing also known as?

Answer 36

sequencing by synthesis.

Question 37

Steps of illumina sequencing?

Answer 37

1. Fragmentation of DNA 2. Immobilisation of DNA fragments – solid support 3. Amplification of immobilised fragments 4. Reversible terminator sequencing – Illumina sequencing