W3 - DNA Sequencing

Question 1

What is Dideoxy sequencing also known as?

Answer 1

Sanger sequencing

Question 2

How is Sanger sequencing similar and different to PCR?

Answer 2

Different:
Some particles cycle to repeat the temperatures but only use a single forward primer. This means amplification is not exponential - so doesn’t create a chain reaction.

Similar:
Also uses a DNA polymerase. But if cycling is performed, a thermostable polymerase would be necessary and are usually used.

Ie. After 30 cycles of Sanger cycling, you would end up with 30 additional molecules of complementary sequence.

Question 3

What are the four stages of Sanger Sequencing?

Answer 3

• Strand separation
  -Annealing Primer
• Extension
• chain termination

If the reaction is cycled, these reactions are repeated.

Question 4

What happens at the strand separation stage?

Answer 4

• Dideoxy and Deoxy nucleotides are added to the DNA duplex.

Question 5

How do you anneal primers?

Answer 5

• A clonal population of identical molecules exist. Could be: A product of PCR, plasmid, sample of genomic DNA
• to design a primer, you must know something about the DNA to be complimentary to a portion of it. Needs to be 5’ to the region we need to sequence.
• anneal /hybridise primer to template strand to form a partially double-stranded structure. (just like PCR, driven by molar excess of primer and competition with renaturation of the template. Can be recognised with DNA polymerase).
• forms initiation complex starts elongation from primer from 3’ OH group.

Question 6

What are the conditions required for extension?

Answer 6

1) A template strand that extend beyond a primer.

2) Free 3’ OH groups on the primer

3) All 4 Deoxy Nucleotide triphosphates ( dATP, dTTP, dCTP, dGTP)

4) Mg2+ ions

Question 7

How does extension occur?

Answer 7

• The primer, dinucleotide is annealed to template strand for base pairing.
• The terminal end of the OH group can react with nucleotide triphosphate to form ester bonds.
• this reaction releases an inorganic pyrophosphate and hydrogen ions.
• The gathering of hydrogen ions gradually acidifies the reaction.

-once the base is added, The polymerase then translocates along molecule to repeat process.

• this process would continue adding basis until the enzyme run out of template, poisoned by acidification, or depletion of nucleotide halting elongation. 

Question 8

What conditions are required for chain termination?

Answer 8

1) A template strand that extend beyond a primer.

2) Free 3’ OH groups on the primer

3) All 4 Deoxy Nucleotide triphosphates ( dATP, dTTP, dCTP, dGTP)

4) All 4 DiDeoxy Nucleotide triphosphates ( ddATP, ddTTP, ddCTP, ddGTP)

5)Mg2+ ions

Question 9

How does chain termination occur?

Answer 9

Achieved by inclusion of the reaction mixture of all four dioxin nucleotide triphosphates (ddATP, ddCTP, ddTTP, ddGTP).

Polymerase will incorporate the deoxy nucleotides with low frequency thus terminate elongation in a low proportion of the elongating strand.

Eg. Adding ddGTP at 1/1000th concentration of dGTP means when polymerase incorporated Guanine, it will have probability of stopping of 1/1000.

Since all bases of dd_TP is present in mixture, the molecule produced by reaction will vary in length depending on when dideoxy nucleotide is incorporated.

Question 10

Why does adding dideoxy neucleotides terminate elongation?

Answer 10

Are dideoxy nucleotide has hydroxyl groups missing one each at the 2’ and 3’ position on the ribose ring. It has a normal 5’ triphospate - so is incorporated by the polymerase all the same.
The polymerase cannot differentiate between this molecules - incorporations due to molar ratio and chance.

Extension of the chain is dependent upon having a free 3’ OH group.

By incorporating a modified nucleotide with a missing OH, we prevent further extension of the Strand.

We have four types of dideoxy neucleotides. To differentiate, we modify each by adding a fluorescent label. This means all terminations will happen at a different wavelength of colour. 

Question 11

How do we sort these molecules to determine the order of the incorporation of nucleotides?

Answer 11

Ordering these molecules by size allows us to determine the sequence of the new strands - analysis to determine the sequence from 5’ end.

Question 12

How do we sort strands by their size?

Answer 12

So suppression can be done by Gel electrophoresis.

For the purpose of the Dideoxy sequencing, we can do this in a capillary - technique is called capillary electrophoresis.

Use this approach because it has a much higher resolution.

Question 13

How does capillary electrophoresis work?

Answer 13

• regular method.
• place a detector at the cathode end and monitor the fluorescence, as population of molecules pass this point, we may reconstitute the sequence.

The fastest ones are shorter = the labelled dideoxy neucleotide is nearer the primer (5’ end of molecule).
As it gets longer, it’s found nearer to 3’ end. This means we can read in the the 5’ to 3’ order.

Question 14

How are the sizes of the strands measured in reality?

Answer 14

Measurement of fluorescence generates a trace and base calling is automated.

Ie. Measuring fluorescence over time to produce graph. - called an electrophrogram. The peaks correspond to individual bases.

This is generated by a computer algorithm performing based calling to provide the sequence such as you see it in the electropherogram.

Question 15

How is deoxy sequencing used in healthcare?

Answer 15

Gold standard confirmatory test For specific genetic mutations in patients with suspected genetic diseases.
- used to confirm all types of mutations: silent, misense, nonsense, truncating, Indel and Mis-Splicing 
- The one exception low-frequency mosaicism. 

Also used to identify HIV haplotypes resistant to anti-retrovirals to determine therapy HAART

Question 16

How is the deoxy sequencing used in research?

Answer 16

• Mammalian and pathogen gene sequencing
• Clone or PCR Amplicon sequencing to confirm a clones sequence or site- directed mutagenesis.
• “Walking” a gene to identify a causative mutation in candidate studies. 
• confirmation of causative variance associated with genetic disease following Association study.