DNA sequencing Flashcards

1
Q

What is dideoxy chain termination also called?

A

→ Sanger Sequencing

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2
Q

Why is Sanger sequencing reliable?

A

→ low error rate

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3
Q

What are the 5 steps in DNA sequencing?

A
Template
       ↓
Enzymatic sequencing 
       ↓
Size separation of products by capillary electrophoresis
       ↓
Detection of reaction products 
        ↓ 
Readout of sequence
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4
Q

What can the two templates be in DNA sequencing?

A

→ A clone (plasmid)

→ Amplicon (PCR product)

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5
Q

What are the differences between PCR and dideoxy sequencing?

A

→ 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

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6
Q

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

A

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

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7
Q

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

A

→ 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

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8
Q

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

A

→ 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

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9
Q

How is pyrophosphate produced in the extension step?

A

→ 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

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10
Q

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

A

→ 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

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11
Q

What are the 5 requirements for chain termination?

A

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

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12
Q

How are molecules of varying length produced?

A

→ 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

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13
Q

Why is the sequence terminated if a dideoxynucleotide is added?

A

→ They do not have an OH group at the bottom

→ the 3’ position does not exist anymore so no more nucleotides can be added

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14
Q

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

A

→ It has a fluorophore attached to it

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

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15
Q

How does DNA polymerase elongate strands?

A

→ 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

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16
Q

What is produced after DNA polymerase elongates the strands?

A

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

17
Q

What is the end goal of the polymerase reaction?

A

→ to have enough of the varying length molecules

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

18
Q

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

A

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

19
Q

How do you find the sequence of the DNA?

A

→ 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

20
Q

How is size separation done?

A

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

21
Q

Where do the fragments move during size separation?

A

→ 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

22
Q

How are the molecules detected?

A

→ the smallest molecule is closest to the 5’ end

→ the largest molecule is closest to the 3’ end

23
Q

What is an electropherogram?

A

→ 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

24
Q

What kind of mutations can dideoxy sequencing confirm ?

A
→ Silent 
→ Missense 
→ Nonsense
→ Truncating 
→ Indel and mis-splicing
25
Q

What mutations can dideoxy sequencing not confirm?

A

→ Low frequency mosaicism

26
Q

What 4 things are DNA sequencing used for?

A

→ 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