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Molecular Biology and Genetics > DNA sequencing > Flashcards

DNA sequencing Flashcards

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1
Q

What did Maxam and Gilbert develop?

A

Sequencing.

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2
Q

When did Maxam and Gilbert develop sequencing?

A

In 1977-80.

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3
Q

How was ‘sequencing’ characterised?

A

The first widely-adopted method for DNA sequencing.

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4
Q

To what was ‘sequencing’ based?

A

On nucleobase-specific partial chemical modification of DNA.

On subsequent cleavage of DNA backbone at sites adjacent to modified nucleotides.

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5
Q

What was DNA Kinase needed about?

A

To add radioactive 32P to the 5’ end of the strand.

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6
Q

Why was DNA Kinase needed to add radioactive 32P to the 5’ end of the strand?

A

To sequence the DNA strand.

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7
Q

How many different reactions wee run on a polyacrylamide gel?

A

4.

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8
Q

Where di DNA sequence read up?

A

Across the 4 lanes of the 4 different reactions.

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9
Q

By what was the Maxam-Gilbert sequencing replaced?

A

By the Sanger method.

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10
Q

What was the function of the Maxam-Gilbert method of DNA sequencing?

A

Destroying each of the 4 bases in separate reaction.

Running the 4 separate reaction on acrylamide gels.

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11
Q

Why was the Maxam & Gilbert method of DNA sequencing running the 4 separate reaction on acrylamide gels?

A

To read off the sequence.

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12
Q

By who was the ‘Sanger sequencing’ developed?

A

Sanger et al.

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13
Q

When was the ‘Sanger sequencing’ developed?

A

In 1977.

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14
Q

What did the ‘Sanger sequencing’ become?

A

The most widely-adopted method for DNA sequencing.

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15
Q

Until when was the ‘Sanger sequencing’ the most widely-adopted method for DNA sequencing?

A

Until 2010.

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16
Q

On what was ‘Sanger sequencing’ based?

A

The incorporation of chain-terminating dideoxy nucleotides (ddNTPs) by DNA polymerase.

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17
Q

What do the ddNTPs lack?

A

The 3’-OH.

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18
Q

What happens when the ddNTPs have been inserted by DNA polymerase?

A

No further elongation occurs.

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19
Q

By what can fluorescent ddNTPs be detected?

A

A laser.

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20
Q

How was the fluorescent ddNTPs characterised originally?

A

Very expensive method.

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21
Q

How is the fluorescent ddNTPs method characterised now?

A

Very cheap.

Efficient procedure.

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22
Q

What does the sequencing mixture contain?

A

Normal dNTPs + a lower concentration of fluorescently-labelled ddNTPs.

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23
Q

What is DNA Polymerase continually adding?

A

dNTPs.

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24
Q

Until when is DNA Polymerase continually adding dNTPs?

A

Until it adds a fluorescent ddNTP.

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25
Q

What does a fluorescent ddNTP stop?

A

Further DNA synthesis.

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26
Q

What happens to the fragments after the sequencing reaction?

A

They are electrophoresed.

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27
Q

Where are the fragments electrophoresed, after the sequencing reaction?

A

In a capillary tube.

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28
Q

By what are the fragments read off in a capillary tube, after the sequencing reaction, and how?

A

By a laser.

One-by-one.

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29
Q

What did the early method of sequencing needed?

A

32P-labelled primers/32P-labelled ddNTPs.

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30
Q

Where were the reactions performed, in the early sequencing method?

A

In separate tubes.

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31
Q

Where were the reactions of sequencing run after they were performed in separate tubes, in the early method of sequencing?

A

On gels.

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32
Q

What is an example of gels where sequencing reactions run on after they are performed in separate tubes?

A

Those used in Maxam & Gilbert sequencing.

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33
Q

From where are DNA sequences determined?

A

Electrophoretograms.

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34
Q

How are the fluorescent signals shown in electrophoretograms?

A

With using different colours for each base.

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35
Q

How many peaks should occur at any time in electrophoretograms?

A

Only one.

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36
Q

Of what are the double peaks the result?

A

Of a mixed DNA template.

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37
Q

What can the mixed DNA template be?

A

Diploid.

Contaminated.

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38
Q

For what is sequencing diploid genomic DNA used?

A

To show if individuals are homozygous/heterozygous at a particular loci.

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39
Q

What are the major benefits of the Sanger sequencing?

A

Cost-effectiveness.
Efficiency.
Reliability.

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40
Q

What reads of sequencing are possible?

A

Long reads up to 800 n.

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41
Q

With what can modifications deal?

A

With highly-repetitive sequences.

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42
Q

What was an ideal method to show the sequence of relatively small fragments of DNA?

A

Over-lapping reads from different primers and different DNA fragments .

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43
Q

Why were the over-lapping reads from different primers, an ideal method to show the sequence of relatively small fragments of DNA?

A

Because they rapidly build up the sequence of a larger region.

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44
Q

What is a PCR-based Sanger sequencing?

A

A very fast way to confirm the sequence of a small section of DNA.

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45
Q

What does a PCR-based Sanger sequencing do?

A

Confirms cloning.

Diagnostics.

46
Q

What does the ‘Next Generation (NGS) sequencing’ include?

A

New techniques.

New technologies.

47
Q

Where are the new techniques and technologies based on?

A

High-throughput approaches with massive parallel sequencing.

48
Q

Why is the ‘Next Generation sequencing’ based on high-throughput approaches with massive parallel sequencing?

A

To generate final sequences of more than 1Mb at a time.

49
Q

How are may techniques aim to show the sequences of lots of small fragments?

A

All at once.

50
Q

In what can the sequences of small fragments combined?

A

In silico.

51
Q

Why can the sequences of small fragments be combined in silico?

A

To generate the sequence of very large fragments.

52
Q

What is the advantage of NGS?

A

In one processing period huge amounts of DNA sequence can be obtained.

53
Q

When is the advantage of NGS only cost-efficient?

A

If near-genome sized sequencing is required.

54
Q

What does high-quality DNA produces when it is prepared and fragmented?

A

Smaller pieces.

55
Q

What does high-quality DNA produce when it is amplified?

A

Greater copies of target DNA.

56
Q

What does an amplification of DNA include?

A

The use of ‘linkers’/’adaptors’.

57
Q

How are the greater copied of target DNA sequenced?

A

By using the by-products of DNA synthesis (PPi).

58
Q

Why are the greater copies of target DNA sequenced by using the by-products of DNA synthesis?

A

To produce light (pyrosequencing).
Addition of fluorescent primer (sequencing-by-ligation).
Change in fluorescent colour.
Production of H+ (sequencing-by-synthesis).

59
Q

When where the number of different versions of pyrosequencing developed?

A

In 1993.

1998.2005.

60
Q

On what does pyrosequencing rely?

A

On light detection.

61
Q

On what is light detection based?

A

On a chain reaction.

62
Q

How is the light detected?

A

By using the pyrophosphate released during DNA synthesis by DNA Polymerase.

63
Q

How is the light produced?

A

By using Sulfurylase and Luciferase.

64
Q

By what are solid array, wells or beads flooded?

A

By each dNTP.

65
Q

Why was the flash of light recorded?

A

To show the DNA sequence.

66
Q

Of what was the bead-based system capable?

A

Of sequencing 400-600 megabases of DNA per 10-hour run.

67
Q

Who developed the ‘Illumina Sequence-by-synthesis sequencing’ first?

A

Solexa.

68
Q

By who was ‘Illumina Sequence-by-synthesis sequencing’ acquired?

A

Illumina.

69
Q

On what was ‘Illumina Sequence-by-synthesis sequencing’ based?

A

On reversible dye-terminators.

70
Q

In what was ‘Illumina Sequence-by-synthesis sequencing’ developed?

A

In a wide range of sequencing-based technologies.

71
Q

Which were some of the sequencing-based-technologies?

A

Transcriptomics.
Methylation profiling.
DNA-protein interactions.

72
Q

What happens to DNA after purification?

A

It is tagged?

73
Q

How is the process of DNA tagging named?

A

Tag mentation.

74
Q

How was DNA tagged?

A

By using transposase.

75
Q

What does transposase cut?

A

It cuts randomly the DNA into short fragments.

76
Q

What is added to either end of cut DNA fragments?

A

Adaptors.

77
Q

Where are the adaptors attached?

A

To a solid surface.

78
Q

What happens to the adaptors attached to DNA fragments after they attach to a solid surface?

A

They are amplified.

79
Q

Why are adaptors amplified?

A

To provide more template DNA.

80
Q

What does DNA Pol add?

A

Fluorescently-labelled dNTPs.

81
Q

How does DNA Pol add fluorescently-labelled dNTPs?

A

With a reversible terminator.

82
Q

How are fluorescently-labelled dNTPs detected?

A

By a laser.

83
Q

What do cycles of removing the terminator and adding new dNTPs provide?

A

Sequence information.

84
Q

What happens in ‘Illumina Sequence-by-synthesis sequencing’?

A

Hundreds of thousands of individually-labelled templates are ‘read’ by laser and are linked to a solid support.

85
Q

What is ‘Ion semiconductor sequencing’?

A

A method of sequencing.

86
Q

On what is the ‘Ion semiconductor sequencing’ based?

A

On the detection of hydrogen ions released during polymerization of DNA.

87
Q

By what are the hydrogen ions detected during polymerization of DNA developed and when?

A

By Ion Torrent Systems Inc.

In 2010.

88
Q

How is the sequencing based on the detection of hydrogen ions released during DNA polymerization named?

A

‘Sequencing by synthesis’.

89
Q

By what is the release of hydrogen ions detected?

A

By an ion-sensitive field-effect transistor (ISFET) ion sensor.

90
Q

What are the major benefits of ion semiconductor sequencing?

A

Rapid sequencing speed.

Low upfront and operating costs.

91
Q

What are the major limitations of ion semiconductor sequencing?

A

Dealing with long repeats of the same base.

Relatively short read lengths.

92
Q

How short are the read lengths of ion semiconductor sequencing?

A

Approximately 400 n per read.

93
Q

What are the ‘ion sequencing steps’?

A

Microwells on a semiconductor chip.

94
Q

What do the micro wells of ion sequencing steps contain?

A

Many copies of one single-stranded template DNA molecules.

95
Q

What happens to the DNA Pol in ion sequencing steps?

A

They are sequentially flooded.

96
Q

How are the DNA Pol sequentially flooded?

A

With unmodified dNTPs.

97
Q

What happens if an introduced dNTP is incorporated into the growing DNA strand by DNA Pol?

A

A H+ ion is released.

98
Q

What does the H+ ion released in the reaction release?

A

The pH of the solution.

99
Q

By what is the change of the pH of the solution detected?

A

By an ISFET sensor under each well.

100
Q

What happens to the unattached dNTPs before the next cycle, when dNTP is introduced?

A

They are washed.

101
Q

What will the DNA Polymerase produce every time a dNTP is successfully added to growing chain of DNA?

A

A H+ ion + pyrophosphate.

102
Q

What do modern high throughput sequencing projects use?

A

NGS technologies.

103
Q

Why do the modern high throughput sequencing projects use NGS technologies?

A

To rapidly produce a draft meeting.

To add reliability to all base pair positions.

104
Q

What does the Ion Torrent produce?

A

Fast drafts with low quality.

105
Q

What does the Illumina produce?

A

Better-quality sequences with short reads.

106
Q

By what is further confirmation of sequences done?

A

By PCR-based Sanger sequencing.

107
Q

What happened to high throughput sequencing costs?

A

They have dramatically fallen.

108
Q

What is it a fact about the use of high throughput sequencing?

A

Very cheap to use through specialist sequencing centres.

109
Q

Where can high throughput sequencing be more efficient than PC-based Sanger sequencing?

A

When there are multiple small samples to analyse NGS.

110
Q

What are the characteristics of High throughput sequencing?

A

Efficient.

Fast.

111
Q

What is it easier to do due to the fact that high throughput sequencing is efficient and fast?

A

To ‘sequence everything’ and then use bioinformatics to see what is in the sequences.

112
Q

For what is sequencing and then bioinformatics easier?

A

For viruses.

Pathogens.

Molecular Biology and Genetics (15 decks)

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