DNA Sequencing

Question 1

How does sequencing by dideoxy chain termination work?

Answer 1

A technique with multiple steps
We have to produce a template
- Often this is done by PCR but could be achieved through other methods
Next we perform a sequencing reaction using DNA polymerase (this step is similar to PCR)
The next step facilitates the determination of the sequencing itself
In this step, separation of the labelled molecules occurs, sorting them out by size and thus length
This is achieved by capillary electrophoresis giving high resolution molecule separation but differ in size by a single base since individual molecules are terminated by a particular dideoxy nucleotide
Determined by the sequence, the original sequence can be reconstructed from the readout

Question 2

Describe the strand separation and annealing primer stage of dideoxy chain termination sequencing

Answer 2

Sequencing Using DNA polymerase
DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides
1. A single stranded oligonucleotide (primer) is bound to the template,
2. The polymerase recognizes the DNA structure, then forms an initiation complex

Question 3

Describe the extension stage of dideoxy chain termination sequencing

Answer 3

Requires:
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
The DNA polymerase recognises the template, initiates elongation and extends the primer
A closer look at the elongating strand: addition of dNTP (base)
The primer’s terminal OH group can react with the phosphate of a nucleotide triphosphate
The reaction forms an ester bonds which releases inorganic pyrophosphate and hydrogen ions thus elongating the strand
As with PCR the reaction releases hydrogen ions and therefore results in an eventual acidification of the reaction
Once the base is successfully added, the polymerase then translocates along the molecule to repeat the process

Question 4

Describe the chain termination stage of dideoxy chain termination sequencing

Answer 4

The reaction became poisoned by acidification or depletion of nucleotides halted elongation.
In which case we’d have no way of determining the sequence which is why the last stage we need is chain termination
Chain termination requires:
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
Chain termination allows us to randomly halt the elongation
This is simply achieved by the inclusion in the reaction mixture of all four dideoxy nucleotide triphosphates
By doing this at low molar ratios of the dideoxy to the deoxy nucleotides, the polymerase will incorporate a dideoxy nucleotide with low frequency and thus terminate elongation in a low proportion of the elongating strands
The reaction mixture consists of billions of strands of DNA so as a consequence we are able to terminate elongation at every position in the template millions times over

Question 5

How do dideoxynucleotides prevent elongation?

Answer 5

Dideoxynucleotides prevent elongation
By incorporating a modified nucleotide with a missing OH we prevent further extension of the strand
But to distinguish between each of the dideoxy nucleotide bases we modify each by adding a fluorescent label

Question 6

How can we separate the DNA strands by size?

Answer 6

Size separation 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

Question 7

How can we determine the sequence after dideoxy chain termination sequencing?

Answer 7

The sequence is determined simply by the direct comparison of the lengths of products terminated by each of the four dideoxy-nucleotides.
The fastest will be the smallest molecule and therefore the base that is closer to the five prime end
This means that after we get all the chains detected we can read the sequence from the 5’ to 3’ end
OR
Sequencing Using DNA dependant DNA polymerase
Measurement of fluorescence generates a trace and base calling is automated
This is an electropherogram
Each peak responds to individual bases
The sequence is created by the computer algorithm using base calling