Sequencing

Question 1

What did Sanger do

Answer 1

-altered the chemistry of the nucleotides to remove the 3’ hydroxyl group
-deoxynucleotide triphosphate is changed to dideoxynuclotide triphospate meaning it no longer has hydroxyl meaning no new bases can be added therefore it gets terminated

Question 2

How can results from Sanger be analysed

Answer 2

Gel electrophoresis which will seperate it due to size

Question 3

Sanger sequencing

Answer 3

4 sequencing reactions performed for each template, each with a different terminator

Radioactive primer or terminator used

After run gel is exposed to film and developed to reveal image

Sequencing reactions run on <1mm polyacrylamide gel cast between two glass plates to separate fragments according to size

Question 4

Combinations of different technologies in Sanger sequencing

Answer 4

• Fluorescent chemistry (Dr Arrowsmith)
• Laser technology
• Imaging
• Data handling and storage
• Computing power

Question 5

Limitations of Sanger sequencing with fluorescence

Answer 5

• Limited to sequencing one sample at a time
• Still gel based
Still incredibly labour intensive – imagine reading 3.2 billion nucleotides!
-expensive technology