10. DNA Sequencing Flashcards

1
Q

What is dideoxy chain termination?

A
  • Often called Sanger Sequencing - This is a technique to seqeunce DNA.
  • Traditional dideoxy nucleotide sequencing by strand termination is a method that uses an enzyme called DNA dependant DNA polymerase to make copies of the complementary strand of a DNA template.
  • It uses a separation step in which the molecles produced are sorted by size and since individual molecules are terminated by a particular dideoxynucleotide determined by the sequence, the original sequence can thus be reconstructed from the readout.
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2
Q

Requirement for amplification of a DNA sequence for sanger sequencing

A
  • Similar to PCR but - some protocols cycle through repeated temperatures BUT only uses a single forward primer - amplification is limited and NOT exponential
  • Uses DNA polymerase - If cycling is performed a thermostable polymerase so would be necessary and is usually used
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3
Q

How does dideoxy chain termination work?

A

1) DNA polymerase makes mutliple copies of the DNA
2) Sorting by size (capillary electrophoresis)
3) Sequential detection of the terminating nucleotide to identify the base
4) Re-constructing the sequence

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4
Q

What are the steps of the reaction of dideoxy chain termination?

A

1) Sequencing reaction
2) Strand separation
3) Annealing primer
4) Extension
5) Chain Termination

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5
Q

Expand on the first 4 steps of the reaction of dideoxy chain termination.

A

• DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides
• A single stranded oligonucleotide (primer) is bound to the template,
the polymerase recognizes the DNA structure, then forms an initiation complex
•Elongation from the 3’ terminus of the primer in a 5’ to 3 ‘ direction.

• Can also cool a denatured template to Tm of the primer to allow annealing.

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6
Q

RECAP: What does DNA dependent DNA polymerase require?

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 (its a cofactor)

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7
Q

What happens at the elongating strand in addition of dNTP?

A
  • Phosphodiester bond between the phosphate on the 5th carbon and the OH group on the 3rd carbon.
  • Release hydrogen ions and inorganic pyrophosphate. (Small amount of hydrogen ions produced compared to in PCR)
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8
Q

What are the requirements of chain termination?

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) All 4 Dideoxy nucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP) ~ Each one labelled with different fluorescent colour to you can tell which one is present
5) Mg2+ ions (its a cofactor)

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9
Q

How an elongating DNA strand terminated?

A
  • Phosphodiester bond between the phosphate on the 5th carbon and the OH group on the 3rd carbon
  • Release hydrogen ions and inorganic pyrophosphate. (Small amount of hydrogen ions produced compared to in PCR)
  • Missing hydroxyl group on the dideoxy prevents further extension - as DNA polymerase will not be able to add more nucleotide to the elongating strand

• The particular dideoxy nucleotide added as a specific fluorescent colour attached to it, so we can tell which one has been added.

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10
Q

Why is the different fluorescent colours important for sequencing?

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.
  • Grouping the different colours and then ordering these molecules by size allows us to determine the sequence of the new strand
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11
Q

Explain how the products of the reaction are separated.

A
  • 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

• Gel electrophoresis is done in a capillary (a fine tube) filled with matrix
nucleotides are attached.

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12
Q

What happens when the sample passes the detector? and how is the sequence determined?

A
  • The different molecules give out fluorescent at different wavelengths and the amount of light increases when they pass the detector. - when they pass through, the peak falls to baseline ~ there is a generation of an electropherogram (a trace)- and capture the light that is emitted and so you can identify which of the 4
  • Measurement of fluorescence generates a trace and base calling is automated
  • The sequence is determined simply by the direct comparison of the lengths of products terminated by each of the four dideoxy-nucleotides.
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13
Q

How is a sequence automated?

A
  • By ABI 3730
  • Samples prepared by dideoxy chain termination on a large scale by robotics
  • Has read the length of up to 900bp and 99.95% accuracy
  • Handles 48 or 96 samples simultaneously
  • More than 1000 samples per day
  • Only performs the separation of labelled DNA and determines the sequence – requires considerable hands on manipulation
  • This technique was used to sequence the human genomes
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14
Q

How is DNA sequencing by dideoxy chain termination used in health?

A
  • Still used, as it is still the gold standard confirmatory test for specific genetic mutations in patients with suspected genetic diseases.
  • Used to confirm all types of mutation (Silent, Misense, Nonsense, Truncating, Indel, and Mis-Splicing) except low frequency mosaicism
  • Identifies HIV haplotypes resistant to anti retrovirals HAART (highly active antiretroviral therapy) - if treatment will fail or not, if so, you need to modify this.
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15
Q

How is DNA sequencing by dideoxy chain termination used in research?

A
  • Mammalian and pathogen gene sequencing
  • Clone or PCR amplicon sequencing to confirm a cloned 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
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