Sanger Sequencing Method

Question 1

Step two of Sanger sequencing

Answer 1

Primer annealing

• > primer annals at 3’ end of template strand
• > up to a maximum of 20 BP long

Question 2

Step three of Sanger sequencing

Answer 2

Extension

• > DNA polymerase assembles new DNA chains using 4 nitrogenous bases (dNTP)
• > DNA polymerase will not work without a primer

Question 3

Step four of Sanger sequencing

Answer 3

Termination
-> reaction mixture contains fluorescent labeled dideoxyribonucleotides (ddNTP) (missing a hydroxyl group on the third carbon) and stops elongation

Question 4

Step five of the Sanger sequence

Answer 4

Gel electrophoresis

• > DNA fragments are run through a gel according to size
• > DNA fragments differ from one base pair so the gel is read from the bottom to the top

Question 5

Step 1 of Sanger sequencing

Answer 5

Double stranded is separated into single stranded using high heat