DNA Sequencing And Dideoxy-chain Termination

Question 1

What is another name for dideoxy-chain termination?

Answer 1

Sanger sequencing

Question 2

What is the read length of dideoxy-chain termination?

Answer 2

900 bps

Question 3

How many samples can a dideoxy-chain termination robot do simultaneously?

Answer 3

48 or 96

Question 4

What are the basic steps of dideoxy-chain termination?

Answer 4

DNA polymerase makes multiple copies of the DNA
They’re sorted by size
Sequential detection of the terminating nucleotide to identify the base
Reconstructing the sequence

Question 5

How is dideoxy-chain termination similar to PCR?

Answer 5

Same protocols and they cycle through repeated temperatures and uses a thermostable DNA polymerase

Question 6

What are the differences between dideoxy-chain termination and PCR?

Answer 6

dideoxy-chain termination only uses a single forward primer and amplification is limited (not exponential)

Question 7

What is the process in dideoxy-chain termination?

Answer 7

Strand separation
Annealing primer
Extension
Chain termination

Question 8

What are the superfluous products of elongation and what do they cause?

Answer 8

Pyrophosphate and hydrogen ions - causes the environment to slowly acidify

Question 9

What things limit elongation?

Answer 9

Enzyme runs out of template, the environment is poisoned by acidification or there is a lack of nucelotides

Question 10

What does chain termination need?

Answer 10

A template strand
A free 3’ OH group on the primer
All four deoxynucleotidetriphosphates and all four dideoxynucleotidetriphosphates
Magnesium ions

Question 11

Why do dideoxynucleotidetriphosphates stop elongation?

Answer 11

They have two hydroxyl groups instead of one hydroxyl and one phosphate, meaning that no more nucleotides can be added

Question 12

Why do the chains vary in length in Sanger sequencing?

Answer 12

Length depends on when the dideoxynucleotide is incorporated into the strand

Question 13

How do you determine the DNA sequence based on the strands in dideoxy chain termination?

Answer 13

Gel electrophoresis - the smallest molecules (closer to 5’ end) move faster than the larger ones (closer to the 3’ end)

Question 14

In practice how is the DNA sequence measured in sanger sequencing?

Answer 14

Forming an electropherogram from the fluorescent signals

Question 15

What are the applications of dideoxy chain termination in healthcare?

Answer 15

It means you can confirm specific gene mutations in patients with genetic diseases and identify antiretroviral resistant HIV

Question 16

What are the applications of dideoxy chain termination in research?

Answer 16

Mammalian and pathogen gene sequencing
Clone or PCR amplicon sequencing to confirm a clones sequence or site-directed mutagenesis
‘Walking’ a gene to identify a causative mutation in candidate gene studies
Confirmation of causative variants associated with genetic disease following association studies