DNA Sequencing

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)

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 (2005 - ) Fast (60Gb or more per day) Parallel sequencing

DNA Sequencing Scramble Class instructions

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

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!!!

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

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

This is the DNA we are going to sequence

This is the primer that will begin the reaction

Annealed primer

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

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.

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

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

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.

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.

Automated Sequencing Results Read sequence in this direction

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

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