1. 6.3 – Manipulating genomes
DNA Sequencing
6.3 – Manipulating genomes

2. Specification Reference

3. Learning Objective Success Criteria
Understand the principles of DNA sequencing and the development of new DNA sequencing techniques.
Describe the developments in DNA sequencing that have occurred in the past – with reference to Sanger sequencing.
Give examples of new techniques that have made sequencing more efficient.
Explain how sequencing allows us to make genome-wide comparisons between species.
Explore the field of synthetic biology and its applications.

4. Starter
23 and me is a service that allows you to send off a saliva sample containing genetic material, and then receive a report outlining “what your DNA says about you.”
You will receive information about:
Do you think this is a good idea?
What if you found out you were at risk of a life-threatening condition?
What if your genetic information was leaked? Or passed to insurance companies?
Can we know too much?

5. Watch the video to find out more about the method.
DNA Research
In 1975, Frederick Sanger, the ‘father of genomics’, developed a method that ultimately allowed scientists to sequence whole genomes.
Sanger’s approach was to use a single strand of DNA as a template for four experiments.
In each experiment, the complementary strand would be synthesised, but the presence of a special, modified nucleotide would terminate it synthesis.
This would result in fragments of DNA being produced of varying lengths.
Watch the video to find out more about the method.

6. Sanger Sequencing
The video describes the modified nucleotides as being fluorescently labelled, but originally they were radioactively labelled.
The smallest fragments are towards the bottom of the gel.
Can you use the diagram to find the base sequence of the DNA strand?
T G A C C A G A T C
This method of sequencing is accurate, but very time-consuming.
Terminator C
Terminator A
Terminator T
Terminator G
Newer, quicker methods based on Sanger’s ideas are now used.
High throughput sequencing has allowed scientists to rapidly sequence whole genomes.

7. What is the base sequence of Suspect 2’s DNA sample?
Which suspect’s DNA has been found at the crime scene? Explain.

8. Pyrosequencing
Pyrosequencing involves synthesising a strand of DNA, one base at a time, that is complementary to the strand being sequenced.
Light is emitted when a base is added, which helps to work out the sequence of bases.
The DNA being sequenced is cut into short pieces, then separated into single strands (ssDNA).
A primer is then added, and the DNA is incubated with:
DNA polymerase
ATP sulfurylase
enzymes
luciferase
apyrase
APS
luciferin
substrates

9. So what happens… light Strand being sequenced Primer
As the triphosphate nucleotide binds, the two extra phosphates are released (pyrophosphate)
If the nucleotide added was not complementary, no light would have been emitted.
Unincorporated nucleotides are degraded by the final enzyme: apyrase.
The reaction will continue when the next nucleotide is added.
The light is detected by a camera.
The amount of light is proportional to the amount of ATP and therefore indicates how many nucleotides were added.
light
oxyluciferin
luciferin
ATP
APS
In the presence of APS, sulfurylase converts pyrophosphate to ATP
One at a time, special activated ‘triphosphate’ nucleotides are added.
In the presence of this ATP, luciferase can convert, luciferin to oxyluciferin, which produces light.

10. Applications of Gene Sequencing
DNA sequencing has allowed for the sequencing entire genomes.
This allows us to:
Make comparisons between species
Explore evolutionary relationships
Explore variation between individuals

11. Predicting Amino Acid Sequences
In year 12, you learnt about protein synthesis.
You learnt that 3 bases (a codon) codes for an amino acid.
If researchers have sequences a genome, they can determine the primary structure of the protein a gene codes for.
All they have to do is use the genetic code.
Eg: AUG AAU GGG CUC codes for:
Met Asn Gly Leu

12. Synthetic Biology
Gene sequencing has allowed for the development of an area called synthetic biology.
It is an area that combines many biological and engineering concepts.
Look at the table on page 220 that outlines some of the examples of synthetic biology.

13. Plenary T C A A G G G C T G G A T T
The figure on the right shows the brightness generated from each activated nucleotide added during high throughput sequencing.
What is the sequence of the nucleotide bases on the length of DNA being sequenced?
T C A A G G G C T G G A T T

14. Learning Objective Success Criteria
Understand the principles of DNA sequencing and the development of new DNA sequencing techniques.
Describe the developments in DNA sequencing that have occurred in the past – with reference to Sanger sequencing.
Give examples of new techniques that have made sequencing more efficient.
Explain how sequencing allows us to make genome-wide comparisons between species.
Explore the field of synthetic biology and its applications.