Sangar sequencing

Question 1

What are the two main methods of DNA sequencing?

Answer 1

Maxam and gilbert - chemical sequencing

Sanger - dideoxynucleotides (labelled nucleotides incorporated into sequence randomly)

Question 2

What are dideoxynucleotides?

Answer 2

Molecules that resemble normal nucleotides but lack the normal hydroxyl group, due to this replication stops as nucleotides have nothing to join onto

Question 3

What does sanger sequencing require?

Answer 3

Multiple copies of single stranded template DNA, a suitable primer, DNA polymerase, pool of nucleotides and a small proportion of dideoxynucleotides labelled with either radioactive or fluorescent dyes

Question 4

How does sanger sequencing work to determine the order of a sequence?

Answer 4

During replication of the template DNA, occasionally a dideoxynucleotide is taken up stopping replication on that piece of DNA, resulting in a mix of DNA lengths. When ran through electrophoresis different bands will correspond to the sizes, with fluorescent labels at the ends of the fragments.

Question 5

What is the difference between PCR and sanger sequencing?

Answer 5

Forward and reverse primers are done in separate tubes

Question 6

How is sanger sequencing prepared?

Answer 6

When preparing the wells they are loaded with DNA, reagents such as dye, primers and water and the wells are spun to ensure DNA and reagents are mixed at the bottom of the well. Samples are centrifuged, precipitated with 95% ethanol, spun again and then drained, then the process is repeated with 70% ethanol. Results in a dry sample that are either analysed immediately or stored in a dark room as light degrades the dyes. Just before sequencing formaldehyde is added to ensure the DNA remains linear

Question 7

How long does it take to run the sequence?

Answer 7

Each sample tray has 96 wells, 1 per sample, and the analysis has the capacity to analyse either 8 or 16 at a time. It can take 4 hours to run 16 samples