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Genomics > DNA Sequencing > Flashcards

DNA Sequencing Flashcards

1
Q

What is a type of DNA sequencing?

A

Dideoxy-chain termination

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2
Q

What is another name for dideoxy-chain termination?

A

Sanger sequencing

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3
Q

When was sanger sequencing developed?

A

In 1977, developed by Nobel Prize winner Fred Sanger

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4
Q

Is Sanger sequencing still used?

A

The method is still used today. The technology has improved, the technique modified and semi-automated. It is a very robust technique with low error rate therefore reliable.

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5
Q

How does automated DNA sequencing by ABI 3730?

A
  • Samples prepared by dideoxy chain termination on a large scale by robotics
  • Has read length of up to 900bp and 99.95% accuracy
  • Handles 48 or 96 samples simultaneously
  • It samples about 1000 samples per day
  • Only performs the separation of labelled DNA and determines the sequence
  • This is used to sequence the human genome producing 23 giga bases of sequence but took 13 years and $2.7 billion to complete
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6
Q

Describe the process of sequencing by dideoxy chain termination

A
  1. Template
  2. Enzymatic Sequencing reaction
  3. Size Separation of products by capillary electrophoresis
  4. Detection of reaction products
  5. Readout of sequence
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7
Q

What happens during enzymatic sequencing reaction?

A

DNA polymerase makes multiple copies of the DNA

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8
Q

How are products sorted by capillary electrophoresis?

A

Sorting by size

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9
Q

How does detection of reaction products work?

A

Sequential Detection of the terminating nucleotide to identify the base

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10
Q

What happens to the sequence at the end of dideoxy chain termination?

A

Re-constructing the sequence

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11
Q

What enzyme does the traditional dideoxy nucleotide sequencing by strand termination method use?

A

It uses an enzyme called DNA dependent DNA polymerase to make copies of the complementary strand of a DNA template.

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12
Q

How is the original sequence reconstructed from the readout?

A

Uses a separation step in which the molecules produced are sorted by size and since individual molecules are terminated by a particular dedoxynucleotide determined by the sequence, the original sequence can thus be reconstructed from the readout.

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13
Q

What is similar between all dideoxy sequencing reaction?

A

Some protocols cycle through repeated temperatures but only uses a single forward primer - amplification is limited and not exponential. It also uses a DNA polymerase if the cycle is performed a thermostable polymerase so would be necessary and is usually used.

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14
Q

How does DNA polymerase start?

A
  1. A single stranded oligonucleotide (primer) is bound to the template
  2. The polymerase recognises the DNA structure, then forms an initiation complex.
  3. This commences elongation from the 3’ terminus of the primer in a 5’ to 3’ direction.
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15
Q

What does DNA dependent DNA polymerase require during the elongation of sanger sequencing?

A
  1. A template strand that extends beyond a primer
  2. Free 3’ OH group on the primer
  3. All 4 Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
  4. Mg2+ ions
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16
Q

What is required for chain termination in sanger sequencing?

A
  1. A template strand that extends a primer forming a partial duplex
  2. Free 3’OH group on the primer
  3. All 4 deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
  4. All 4 dideoxy nucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP)
  5. Mg2+ ions
17
Q

Explain elongation in sanger sequencing

A

From the 5’ prime end, there is elongation in the 5’ to 3’ direction. At the 3’ OH end, a H+ and inorganic pyrophosphate

18
Q

How is DNA elongation terminated?

A

By the addition of dideoxynucleotide

19
Q

How do dideoxynucleotides prevent elongation?

A

The 3’OH group is missing which prevents the connection between H+ and pyrophosphate.

20
Q

Summarise the sequencing using DNA polymerase

A
  1. The polymerase commences elongation from the 3’ terminus.
  2. As the enzyme encounters a particular nucleotide in the sequence it picks out and incorporates a complementary nucleotide into the elongating strand.
21
Q

Why is it important to have all ddNTP?

A
  • Products where a ddCTP is incorporated therefore represent all positions within the sequence where a “cytosine” occurs.
  • Since all four labelled dideoxy nucleotides are present in the reaction the population of molecules produced represent all possible positions in the sequence from the same point to the end.
22
Q

What does ordering these molecules by size allow?

A

It allows us to determine the sequence of the new strand

23
Q

How does size separation happen? Explain how it happens

A

It can be done by gel electrophoresis.

  • The nucleic acid passes through a gel matrix by applying a voltage across two electrodes.
  • Negatively charged nucleic acid migrates towards the positive electrode.
  • The matrix retards the molecules according to their size
  • Those that are larger are retarded to a greater extent and as a consequence move through the matrix more slowly
24
Q

How is the sequence determined?

A

The sequence is determined simply by the direct comparison of the lengths of products terminated by each of the four dideoxy-nucleotides.

25
Q

What does the DNA dependent DNA polymerase generate?

A

Measurement of fluorescence generates a trace and base calling is automated

26
Q

When is DNA sequencing by dideoxy chain termination used?

A
  • Health

- In reasearch

27
Q

When is Sanger sequencing used in health?

A
  • Confirmatory test for specific genetic mutations in patients with suspected genetic
    diseases.
  • Identifying HIV haplotypes resistant to anti-retrovirals HAART
28
Q

What types of mutations does Sanger sequencing identify? And what is the one expection?

A

Silent, Missense, Nonsense, Truncating, Indel, and mis-splicing.
The one exception is low frequency mosaicism

29
Q

When is Sanger Sequencing used in research?

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 association study

Genomics (19 decks)

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