DNA Sequencing

Question 1

List the 5 components needed for Sanger Sequencing

Answer 1

Clean template
short DNA primer
high fidelity primer
unlabeled dntps
fluorescent labelled dntps

Question 2

List the steps of Sanger sequencing

Answer 2

Denaturation
Annealing
Extension
Termination

Question 3

Causes of “blobs” in electropherograms

Answer 3

poor cleanup of sequencing rxn

Question 4

Causes of “shouldering”

Answer 4

Capillary array needs to be replaced
Overloaded sample
Homopolymeric region in sample (Stutter = rpt C)

Question 5

Causes of double peaks (at beginning of sequence)

Answer 5

PCR product used for sequencing

ore than 1 PCR product in rxn

Question 6

What is pyrosequencing?

Question 7

When is pyrosequencing used in favor of sanger sequencing

Answer 7

sequencing methylated DNA

Question 8

Differentiate: Chemiluminescence vs fluorescence

Question 9

Describe the Pyrosequencing workflow

Answer 9

Extraction
PCR
Sample prep
Pyrosequence
Analysis

Question 10

Describe dispensation in

Answer 10

order of nucleotide addition to pyrosequencing rxn

Question 11

List the components needed for Pyro-Sequencing

Question 12

What is the result of bisulphite incubation?

Answer 12

deamination of unmethylated cytosine to uracil

Question 13

Describe the workflow of NGS/Illumina

Question 14

Roles of Adapters

Question 15

List and describe the 5 steps of Illumina sequencing

Answer 15

Fragmentation
End repair and phosphorylation
Adenylation
Ligation
(optional) Amplification

Question 16

How is library molar conversion done?

Answer 16

( X ng/uL * 10^6 ) / (660 * library size bp) = Library concentration in nM