DNA sequencing

Question 1

What does each Sanger sequencing reaction mixture contain?

Answer 1

• Primer
• DNA polymerase
• dNTPs
• One of the four ddNTPs

Question 2

In Sanger sequencing, the reaction mixtures are separated by ddNTP. True or false?

Answer 2

True.

Question 3

In Sanger sequencing, all dNTPs are added to all reactions. True or false?

Answer 3

True.

Question 4

Sanger sequencing ends with gel electrophoresis to visualise the sequence. True or false?

Answer 4

True.

Question 5

In automated Sanger sequencing, there are four reactions (one for each ddNTP). True or false?

Answer 5

False.

Question 6

What is the central difference of automated Sanger sequencing?

Answer 6

The bases of the ddNTPs are fluorescently labelled.

Question 7

What type of electrophoresis does automated Sanger sequencing use?

Answer 7

Capillary gel electrophoresis.

Question 8

What is another term for sequencing trace?

Answer 8

Electropherogram.

Question 9

At what length does Sanger sequencing begin to lose reliability?

Answer 9

700-1,000 bp.

Question 10

What are three weaknesses of Sanger sequencing?

Answer 10

1. Poor sequencing at primer and end.
2. Cost-inefficient.
3. Struggles with repeat base calls.

Question 11

Does Sanger sequencing require knowledge of the target sequence?

Answer 11

Yes.

Question 12

Automated Sanger sequencing uses fluorophore-tagged primers to sequence DNA. True or false?

Answer 12

False.

Question 13

What are flow cells?

Answer 13

Glass slides containing a series of primer lawns.

Question 14

What are primer lawns?

Answer 14

Localised areas of chemically-attached oligonucleotides.

Question 15

What are the two steps of sample preparation for NGS?

Answer 15

1. Fragmentation of the DNA strand.
2. Attachment of adaptors.

Question 16

DNA adaptors are complementary to the primer lawns. True or false?

Answer 16

True.

Question 17

What is the first step of cluster generation in NGS?

Answer 17

Hybridisation of the adaptor sequences to the primer lawn.

Question 18

How many different primers are there on the lawn?

Answer 18

Two.

Question 19

In NGS, which DNA remains after fragment hybridisation and extension: the template or the clone?

Answer 19

The clone.

Question 20

The 3’ end of the clone bends to bind the other oligonucleotide on the primer lawn. True or false?

Answer 20

True.

Question 21

The bending and extension of the DNA to the second oligonucleotide is referred to as…

Answer 21

Bridge amplification.

Question 22

After bridge amplification, we have two forward sequences of the DNA. True or false?

Answer 22

False.

Question 23

After bridge amplification, what do we call the regions of DNA strands on the flow cell?

Answer 23

Clusters.

Question 24

How are we left with just forward strands at the end of cluster generation?

Answer 24

Enzymes cleave the reverse strands away.

Question 25

NGS uses ddNTPs. True or false?

Answer 25

False.

Question 26

What is at the 3'-OH of modified dNTPs in NGS?

Answer 26

A blocker molecule.

Question 27

In NGS, is the sequencing strand extended towards or away from the flow cell?

Answer 27

Towards.

Question 28

Is another primer needed to begin sequencing after cluster generation?

Answer 28

Yes.

Question 29

What two things must be removed after every dNTP in NGS?

Answer 29

1. Fluorophore. 2. 3' blocker molecule.

Question 30

In sequence assembly, what do we often do for repeat regions?

Answer 30

Leave a gap.

Question 31

Define paired end sequencing.

Answer 31

Two reads of the same sequence, a 5'-3' and a 3'-5'.

Question 32

What characterises DNA used in mate-pair sequencing?

Answer 32

Much longer fragment sizes.

Question 33

What are three applications of NGS?

Answer 33

1. Whole genome sequencing. 2. Single-cell sequencing. 3. RNA sequencing.

Question 34

Is NGS low or high throughput?

Answer 34

High.

Question 35

What is a key reason NGS is great at genome/single-cell sequencing?

Answer 35

Can detect SNPs with high accuracy.