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Genomics > DNA Sequencing > Flashcards

DNA Sequencing Flashcards

1
Q

What is another name for Sanger Sequencing?

A

Dideoxy chain termination

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2
Q

Why is Sanger sequencing so reliable?

A

Very robust – with low error rate

Hence it is a “gold standard” technique - used as a comparative for other techniques

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3
Q

Who discovered Sanger sequencing?

A

Method developed by Nobel Prize Winner Fred Sanger to sequence DNA in 1977
Technology has improved, the technique modified and semi-automated

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4
Q

How are samples prepped for use by the ABI 3730?

A

samples prepared by dideoxy chain termination on a large scale by robotics

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5
Q

How many samples are processed by ABI 3730?

A

Reads length of up to 900bp & 99.95% accuracy
Handles 48 or 96 Samples simultaneously
>1000 samples per day

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6
Q

How does DNA separation occur via the ABI 3730?

A

But only performs the separation of labelled DNA & determines the sequence – requires considerable hands on manipulation

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7
Q

What role did the ABI 3730 play in sequencing human genome?

A

Technique was used to sequence the Human Genome
Produced 23 giga bases of sequence
But took 13 years & $2.7 bn to complete

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8
Q

Outline the Dideoxy chain termination steps

A
  1. Template used in a sequencing reaction using DNA
    Polymerase to produce multiple copies of the
    complementary strand
  2. Products separated based on size using capillary
    electrophoresis
  3. Sequential detection of terminating nucleotide to
    identify the base
  4. Readout of sequence reconstructed into base sequence
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9
Q

What template is used in Dideoxy chain termination?

A

Template : either a clone (e.g. plasmid) or a PCR product Required in:

  • High degree of purity
  • Large amounts
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10
Q

How are products detected in dideoxychain termination?

A

Individual molecules are terminated by a particular dideoxynucleotide determined by the sequence, so the original sequence can be detected and reconstructed from the readout

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11
Q

How is dideoxy chain termination similar to PCR?

A

Some protocols cycle through repeated temperatures

It also uses a DNA polymerase

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12
Q

How does PCR and dideoxy chain termination differ?

A

Dideoxy chain amplification is limited and NOT exponential

Dideoxy chain termination uses a single forward primer

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13
Q

When is DNA polymerase used in Sanger sequencing?

A

If cycling is performed a thermostable polymerase so would be necessary and is usually used

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14
Q

Outline how sequencing is carried out using DNA Polymerase

A

DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides

  1. A single stranded oligonucleotide (primer) is bound to
    template
  2. Polymerase recognizes DNA structure, forming an
    initiation complex
  3. Commences elongation from 3’ terminus of primer in a
    5’ to 3 ‘ direction
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15
Q

What are the requirements of DNA Dependent DNA polymerase?

A 
Requires: 
- Template strand that extends beyond a primer 
- Free 3’ OH group on primer
- All 4 Deoxynucleotide triphosphates 
  (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions
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16
Q

Describe the structure of the elongating DNA Strand

A

5’ end - 5’ Carbon is attached to triphosphate
Phosphodiester bond forms backbone of DNA molecule
Base has a hydroxyl group at 3’C position allowing the formation of another phosphodiester bond with the added base

17
Q

What are the byproducts of the elongation strand?

A

=> terminal phosphates are released producing inorganic pyrophosphate

=> hydrogen released forming H+ ions

=> during PCR release of H+ causes acidification of the reaction

18
Q

Why does acidification not occur during Sanger sequencing?

A

During Sanger sequencing a relatively low number of H+ ions are released ∴ acidification doesn’t occur, whereas PCR is exponential so produces a lot more H+

19
Q

What are the requirements of dideoxy chain sequencing?

A 
- Template strand that extends a primer forming a partial 
  duplex
- Free 3’ OH group on the primer
- All 4 Deoxynucleotide triphosphates 
  (dATP, dGTP, dCTP, dTTP)
- All 4 Dideoxy nucleotide triphosphates 
  (ddATP, ddGTP, ddCTP, ddTTP)
- Mg2+ ions`
20
Q

How is DNA elongation terminated?

A

DNA elongation is terminated by addition of dideoxynucleotide

21
Q

How does the addition of a dideoxynucleotide terminate elongation?

A

No OH’ on 3’C on ribose ring so elongation can’t occur anymore once dideoxynucleotide has been added

22
Q

What feature enables dideoxynucleotides to be identified?

A

Each dideoxynucleotide suppresses a different wavelength and so can be detected by fluorescent markers

23
Q

How does sequencing occur in the presence of dideoxynucleotides?

A

DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides

  1. 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.
  3. If a dideoxy nucleotide is incorporated into the strand,
    then elongation is terminated otherwise elongation
    continues
24
Q

How big are the reaction products produced via Sanger sequencing?

A

The reaction products vary in length terminated by ddNTP

25
Q

Describe the positions of the ddNTPs within the reaction products

A

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.

Products where a ddCTP is incorporated therefore represent all positions within the sequence where a “Cytosine” occurs

26
Q

How is the sequence of the new strand determined?

A

Ordering these molecules by size allows us to determine the sequence of the new strand

27
Q

What method is used to separate molecules by size?

A

size separation can be done by gel electrophoresis

28
Q

Outline the method of gel electrophoresis

A
  1. The nucleic acid passes through a gel matrix by
    applying a voltage across two electrodes
  2. Negatively charged nucleic acid migrates towards the
    positive electrode.
  3. The matrix retards the molecules according to their size
  4. Those that are larger are retarded to a greater extent
    and as a consequence move through matrix more
    slowly
29
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.

30
Q

How does size effect the position of the molecules along the strand?

A

Smallest molecule is closest to the 5’ end and the largest molecule is closest to the 3’ end

31
Q

How does fluorescence allow us to determine a sequence?

A

Measurement of fluorescence generates a trace and base calling is automated

32
Q

What are the uses of dideoxy chain termination in healthcare?

A

Used to confirm all types of mutation
- Silent, Missense, Nonsense, Truncating, Indel, and Mis-
Splicing
- the one exception low frequency mosaicism
- Identifying HIV haplotypes resistant to anti-retrovirals
HAART

33
Q

What is the significance of Sanger sequencing in healthcare?

A

Gold standard confirmatory test for specific genetic mutations in patients with suspected genetic diseases

34
Q

Outline the research uses of Sanger sequencing

A
  • Mammalian and Pathogen Gene sequencing
  • Clone / 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 (20 decks)

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