1. DNA Sequencing

2. DNA Sequencing Sanger sequencing (1977) Invented by Frederick Sanger
Provided a way to sequence DNA from any organism using the stages of the PCR
Works best with small fragments
Even when automated (1987), still very slow (up to 500kb per day)

3. DNA Sequencing Human Genome Project (1990 - 2003)
Attempt (successful!) to sequence the entire human genome (~32Gb)
Led to development of improved technology for sequencing
Next-generation sequencing ( )
Fast (60Gb or more per day)
Parallel sequencing

4. DNA Sequencing Scramble
Class instructions

5. In this lesson DNA sequencing is a powerful method for analyzing DNA
Ratio of chain terminators (ddNTPs) is important
Importance of complementary base pairing
DNA polymerase only works in the 5’- 3’ direction
Electrophoresis separates DNA fragments by size
Chain terminators stop the reaction & visualize the result
Why fluorescent labels are used more than radioisotopes
Software (bioinformatics) is used to line up sequences

6. The devil is in the detail!
The 5’ prime and 3’ prime ends of the bases must be round the right way!
IMPORTANT:
Do not take bases apart!!!

7. Correct base pairing is critical!
Green (Guanine) pairs with yellow (Cytosine)
Blue (Adenine) pairs with orange (Thymine)

8. Bases with white sugars are CHAIN TERMINATORS or dideoxynucleotides (ddNTP)

9. This is the DNA we are going to sequence

10. This is the primer that will begin the reaction

11. Annealed primer

12. Sanger Sequencing Original method of sequencing
Chain terminating nucleotides are radioactively labeled
Sequencing takes place in four separate tubes, each with one ddNTP (A, T, G or C)
All four results are then run in four separate lanes on a gel, separated by size
Each reaction tube would contain:
- DNA for sequencing - all 4 types of nucleotides
- one type of ddNTP - DNA polymerase
- primers

13. The activity Denature the two strands and anneal the primer:
Randomly select a nucleotide from the pot until it is the correct colour and add it in the 5’ to 3’ direction.
Repeat until you reach a ddNTP, then detach your chain and pass the original DNA to the next student.
Repeat this for each reaction tube.

14. Sanger Sequencing Result - Electrophoresis
Line up products in 4 columns – one for each base:
Read sequence in this direction

15. Read sequence in this direction
How an electrophoresis gel would look; the actual sequence is complementary to this: G A T C
Read sequence in this direction

16. The activity
Each student takes it in turns to synthesise a new strand.
Randomly select a nucleotide from the pot.
Add them in the 5’ to 3’ direction.
If you pull out a ddNTP, your chain is finished and it is the next student’s turn.

17. The result - electrophoresis
Line up all the fragments from longest to smallest in one column
Read the sequence from the smallest fragment upwards; again, the actual sequence will be complementary to this.

18. Automated Sequencing Results
Read sequence in this direction

19. Electrophoresis gel is read by a laser - printout produced
Read sequence in this direction

20. The difference between two methods
Original Sanger method requires four lanes because all fragments are the same colour.
Radioactive labels = hazardous
Automated sequencing can be run in one lane because each fragment is coloured differently.
Fluorescent labels = safe