DNA Sequencing

Question 1

Describe how DNA sequencing has become automated.

Answer 1

A machine commonly used is the ABI 3730.

The samples are prepared by dideoxy chain termination on a large scale by robotics. It has a read length of up to 900 base pairs and 99.95% accuracy.
It handles 48 or 96 samples simultaneously, and can go through more than 1000 samples a day.

But, it only performs the separation of labelled DNA and determines the sequence - it requires considerable hands-on manipulation.

Question 2

Define dideoxy chain termination.

Answer 2

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 molecules 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.

Question 3

What are the steps in dideoxy chain termination?

Answer 3

1. Generating a Template:
   This can either be a clone (plasmid) or an amplicon (from PCR).
2. Sequencing Reaction:
   DNA Polymerase makes multiple copies of the template.
3. Separation on size:
   This is done using capillary electrophoresis.
4. Detection of Reaction Particles:
   Here, sequential detection of the terminating nucleotide to identify the base occurs.
5. Readout of the sequence:
   This is where the sequence is reconstructed.

Question 4

How is dideoxy chain termination different to PCR?

Answer 4

The dideoxy sequencing reaction is similar to some protocols in PCR, such as that they cycle through repeated temperatures. However, dedioxy sequencing only uses a single forward primer - this means that amplification is limited (linear) and not exponential.

They both use a DNA Polymerase (if cycling is performed, a thermostable polymerase would be necessary).

Question 5

What are the different steps in the sequencing reaction in dideoxy chain termination?

Answer 5

1. Strand separation
2. Annealing primer
3. Extension
4. Chain Termination

Question 6

Expand on the first two steps of the sequencing reaction.

Answer 6

The first two steps are strand separation and annealing primer.

The DNA is mixed with the reaction components and a labelled, single stranded nucleotide (?) is bound to the template. The polymerase recognises the DNA and then forms an initiation complex and commences enlongation from the 3’ terminus of the primer in a 5’ to 3’ direction.

Question 7

Expand on the third step of the sequencing reaction.

Answer 7

The third step of the sequencing reaction is extension.

This occurs by the action of DNA polymerase. It requires:

• a template strand that extends beyond a primer
• free 3’ OH group on the primer
• all 4 deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
• Mg2+ ions

Question 8

Expand on the fourth step of the sequencing reaction.

Answer 8

The fourth step of the sequencing reaction is chain termination.

DNA elongation is terminated by the addition of a dideoxynucleotide (ddATP, ddGTP, ddCTP, ddTTP). Dideoxynucleotides prevent elongation.

As the enzyme encounters a particular nucleotide (e.g. guanine) in the sequence, it picks out a complementary cytosine and incorporates it into the elongating strand. However, the reaction contains both dCTP and ddCTP; if a dideoxy molecule is incorporated into the strand, elongation is terminated, but if a deoxy molecule is incorporated, elongation continues.

Question 9

Explain the implications of adding the dideoxy molecules into the sequencing reaction.

Answer 9

Products where a ddCTP (eg) is incorporated 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 produces represent all possible reactions in the sequence from the same point to the end.

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

Question 10

Describe the size separation by gel electrophoresis.

Answer 10

The nucleic acid passes through a gel matrix by applying a voltage across two electrodes.
Negatively charged nucleic acid will migrate 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.

Question 11

How is the sequence determined after it’s size separation?

Answer 11

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

Question 12

What are the implications of DNA sequencing by dideoxy chain termination in health?

Answer 12

Today, dideoxy sequencing is still the gold standard confirmatory test for specific genetic mutations in patients with suspected genetic diseases.

They are used to confirm all types of mutation:
- silent
- misense
- nonsense
- truncating
- indel
- missplicing
[the only exception is low frequency mosaicism]

It is also used in identifying HIV haplotypes that are resistane to anti-retrovirals HAART (this helps us decide if we need to adjust the treatment for certain patients, or to predict if they will fail the treatment altogether).

Question 13

What are the implications of DNA sequencing by dideoxy chain termination in research?

Answer 13

• mammalian and pathogen gene sequencing
• clone or PCR aplicon sequencing (to confirm a clone’s 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