DNA Sequencing

Question 1

What is dideoxy chain termination also called?

Answer 1

→ Sanger Sequencing

Question 2

Why is Sanger sequencing reliable?

Answer 2

→ low error rate

Question 3

What are the 5 steps in DNA sequencing?

Answer 3

Template
↓

Enzymatic sequencing
↓
Size separation of products by capillary electrophoresis
↓
Detection of reaction products
↓
Readout of sequence

Question 4

What can the two templates be in DNA sequencing?

Answer 4

→ A clone (plasmid)

→ Amplicon (PCR product)

Question 5

What are the differences between PCR and dideoxy sequencing?

Answer 5

→ Cycles through repeated temperatures but only uses a single forward primer
→ Amplification is limited (linear) and not exponential

→ Linear = 25 rounds - 25 molecules
→ it uses DNA polymerase

Question 6

What is the first step of the sequencing reaction and how does this occur?

Answer 6

→ A single stranded oligonucleotide is bound to the template DNA
→ The polymerase recognizes the DNA structure and forms an initiation complex

Question 7

What kind of DNA do you have to perform Sanger sequencing with?

Answer 7

→ a piece of DNA of which part of the sequence is already known
→ The primers have to be complementary to part of the sequence for them to bind

Question 8

What are the 4 requirements for the DNA polymerase extension step?

Answer 8

→ A template strand that extends beyond a primer
→ A free 3’ OH group on the primer

→ All 4 deoxynucleotide triphosphates (dATP, dGTP, dTTP, dCTP)
→ Mg2+ ions

Question 9

How is pyrophosphate produced in the extension step?

Answer 9

→ The 3’ end of the molecule has an OH group
→ This is available to form a phosphodiester bonds with a nucleotide that is being added

→ The terminal phosphate is released
→ This produces inorganic pyrophosphate and H+ is released

Question 10

Why does acidification of the reaction not occur in Sanger sequencing?

Answer 10

→ in PCR the release of the H+ ions causes acidification

→ In Sanger sequencing there is a small amount of H+ produced because there are less cycles

Question 11

What are the 5 requirements for chain termination?

Answer 11

→ All 4 dideoxynucleotide triphosphates ( ddATP, ddGTP, ddCTP, ddTTP)

Question 12

How are molecules of varying length produced?

Answer 12

→ Each dideoxynucleotide is labelled with a different fluorescent tag
→ The polymerase cannot differentiate between a deoxy and a dideoxy nucleotide

→ if the polymerase incorporates a dideoxynucleotide the chain will end
→ depending on when the dideoxynucleotide is added the chain lengths will differ

Question 13

Why is the sequence terminated if a dideoxynucleotide is added?

Answer 13

→ has two hydroxyl groups missing one each at the 2’ and 3’ positions of the ribose ring.
But as it has a normal 5’ triphosphate it may be incorporated by the polymerase all the same

→ the 3’ position does not exist anymore so no more nucleotides cannot be added and a phosphodiester bond can’t be made.

Question 14

How can you tell which dideoxynucleotide was incorporated into the sequence?

Answer 14

→ It has a fluorophore attached to it

→ you can tell which nucleotide was incorporated by the fluorescent color that it gives off

Question 15

How does DNA polymerase elongate strands?

Answer 15

→ The polymerase starts elongation from the 3’ terminus
→ As the enzyme encounters a particular nucleotide in the sequence it picks out and incorporates a complementary nucleotide in the elongating strand

Question 16

What is produced after DNA polymerase elongates the strands?

Answer 16

→ A variety of molecules that terminate at different positions within the elongating strand marked by the fluorescence of the dideoxynucleotide

Question 17

What is the end goal of the polymerase reaction?

Answer 17

→ to have enough of the varying length molecules

→ to know that you have terminated every single position within the unknown sequence

Question 18

How do you know where a cytosine occurs in the sequence?

Answer 18

→ Products where a ddCTP is incorporated represent all positions within a sequence where cytosine occurs

Question 19

How do you find the sequence of the DNA?

Answer 19

→ All 4 labelled dideoxy nucleotides are present in the reaction
→ the population of molecules of varying length represent all the possible positions in the sequence from the same point to the end

→ if the molecules are sorted by length the new sequence can be determined

Question 20

How is size separation done?

Answer 20

→ The nucleic acid passes through a gel matrix by applying a voltage across two electrodes

Question 21

Where do the fragments move during size separation?

Answer 21

→ the negatively charged nucleic acid migrate towards the positive electrode
→ the larger fragments are retarded to a greater extent and as a consequence move through the matrix more slowly

Question 22

How are the molecules detected?

Answer 22

→ the smallest molecule is closest to the 5’ end, nearer the primer
→ the largest molecule is closest to the 3’ end

Question 23

What is an electropherogram?

Answer 23

→ It represents the amount of fluorescence over time
→ The left has molecules that are closer to the 5’ end

→ The right has molecules that are closer to the 3’ end

Question 24

What kind of mutations can dideoxy sequencing confirm ?

Answer 24

→ Silent
→ Missense

→ Nonsense
→ Truncating
→ Indel and mis-splicing

Question 25

What mutations can dideoxy sequencing not confirm?

Answer 25

→ Low frequency mosaicism

example if a mutation occurred in a cell in the early stage of embryo development and did not originate in one of the gametes

Question 26

What 4 things are DNA sequencing used for?

Answer 26

→ 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 an association study

Question 27

Why does Sanger sequencing use only a single forward primer?

Answer 27

means amplification thus is limited and NOT exponential because the complementary product of the reaction does not act as a template for subsequent rounds as in PCR