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Molecular Biology > DNA Sequencing > Flashcards

DNA Sequencing Flashcards

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1
Q

What is DNA sequencing?

A

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA.

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2
Q

What is the basic principle of DNA sequencing?

A

The process of DNA sequencing involves both the determination of the unknown bases in the strands of DNA and the sequence in which they occur.

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3
Q

What do you need for DNA sequencing to work?

A

To sequence a DNA molecule a purified single strand of the DNA is required.

Also, for DNA sequencing to work, the identity of the bases in a short sequence has to be known.

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4
Q

How many stages of DNA sequencing are there and what are they?

A

There are four stages: Preparation of the reaction mixtures, DNA synthesis in the Reaction Mixtures, Gel Electrophoresis and Autoradiography.

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5
Q

What is needed in step one of DNA sequencing?

A

Single-stranded DNA with unknown sequence, Radioactively labelled primer, DNA polymerase and Normal Nucleotides

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6
Q

What is added to each of the four reaction mixtures?

A

A specific dideoxynucleotide is added (one type of ddNTP per tube)

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7
Q

What are the four dideoxynucletides?

A

ddATP, ddTTP. ddCTP and ddGTP

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8
Q

What happens during step two of DNA sequencing?

A

Primer binds to the template sequence

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9
Q

How does the primer bind to the template strand?

A

Compliments the known sequence & therefore can bind to it and is Radioactively labelled

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10
Q

What happens during step three of DNA sequencing?

A

DNA polymerase can begin to synthesise DNA, because of the presence of the primer

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11
Q

Can the DNA polymerase synthesise DNA without a primer?

A

No

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12
Q

What happens during step four of DNA sequencing?

A

DNA polymerase uses the template strand to synthesise a new DNA sequence by inserting complementary dNTPs

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13
Q

What happens during step five of DNA sequencing?

A

DNA polymerase continues synthesising DNA with dNTPs

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14
Q

What happens if DNA Polymerase incorporates the ddNTP present in the reaction tube?

A

Synthesis of the new DNA will be terminated

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15
Q

Why does the synthesis stop if a dideoxynucleotide is added?

A

If a ddNTP is added to a growing DNA strand, the chain is not extended any further because the free 3′ OH group needed to add another nucleotide is not available.

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16
Q

What does the length of the new DNA fragments synthesised depend on?

A

The template DNA sequence

17
Q

How many fragments are there in ddATP reaction mixtures?

A

3

18
Q

How many fragments are there in ddTTP reaction mixtures?

A

1

19
Q

How many fragments are there in ddCTP reaction mixtures?

A

2

20
Q

How many fragments are there in ddGTP reaction mixtures?

A

3

21
Q

What is Gel electrophoresis?

A

Gel electrophoresis is a technique which can separate charged molecules on the basis of their size

22
Q

What happens during autoradiography?

A

The fragments on the gel can not be visualised directly. To permit visualisation a piece of X-ray film is laid on top of the gel.

23
Q

What is Sanger sequencing?

A

Sanger sequencing is the utilization of dideoxynucleotide to allow base determination

24
Q

What are the pros and cons of Sanger sequencing?

A

Pros: Simple, fast and accurate (accuracy = 99.9%)

Cons: Only able to sequence short fragments (300 – 1000 bp), Only able to determine one sequence at a time

25
Q

What are the pros of Next Generation sequencing?

A
  • High-throughput sequencing
  • Fully automated, reliable and accurate
  • Large numbers of samples can be analysed in a short time
  • Allows parallel sequencing
26
Q

What is pyrosequencing?

A

The method is based on a chemiluminescent enzymatic reaction, which is triggered when a molecular recognition event occurs

The method allows the sequencing of a single strand of DNA by synthesizing the complementary strand along it

Each time a nucleotide, A, C, G or T is incorporated into the growing chain a cascade of enzymatic reactions is triggered which results in a light signal

Molecular Biology (5 decks)

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