L14 - DNA sequencing

Question 1

What is electrophoresis?

How does it work?

Answer 1

Technique used to separate DNA based on its size –> DNA electrophoresis / agarose gel electrophoresis

• Samples loaded into the wells
• Electric current causes migration of samples. DNA is negative so it moves towards the positive electrode
• Small fragments move more quickly; large fragments more slowly
• Visualise using a dye – ethidium bromide/Midori Green (safer alternatives now exist) – The dye intercalates into DNA and fluoresces under UV light.

Question 2

What are the basics of Sanger sequence/chain termination? how does it work?

Answer 2

Sanger sequencing is a method used to determine a portion of the nucleotide sequence of an individual’s genome.

The last base is found by…using different ddNTPs (dideoxynucleotides).

To know the entire sequence use both ddNTP + “normal” dNTPs (usually an excess of these). Millions of products in our sequencing reaction. Each will incorporate the ddNTP in a different place.

To know how long the DNA molecule is…Gel electrophoresis – discriminate between DNA molecules by size.