Lecture 21

Question 1

What does sequencing a gene allow for?

Answer 1

Sequencing a gene allows us to determine the organisation of the fene, find our what the gene encodes, the function of the RNA or protein it encodes and we can compare the sequence with different organisms.

Question 2

How is dideoxy sequencing of DNA done?

Answer 2

dideoxy sequencing of DNA is done using dye-labeled terminators, this is done by seperating the DNA strands, we then anneal the primer and extend with 4 dideoxy nucleotides (one of each type) which each has a different fluorescent dye, these are known as terminator bases. This is done in capillary tubes filled with capillary gel and then electrophoresis is done to push the DNA sequences along (further if smaller fragments). The order of the DNA can then be read from either end by converting the colours to letters.

Question 3

What does dideoxy sequencing require?

Answer 3

Dideoxy DNA sequencing requires: primer, free DNA bases, terminator bases and DNA polymerase.

Question 4

What are the two strategies for seguencing genomes via cloning?

Answer 4

Sequencing genomes via cloning can be done by ordered clone sequencing or whole genome shotgun sequencing.

Question 5

How is ordered clone sequencing done?

Answer 5

Ordered clone sequencing is done by:

1. Order large-insert clones by overlapping fingerprints to create a physical map
2. select clones with minimal overlap
3. divide into subclones
4. Sequence subclones
5. Assemble subclones.

Question 6

How is whole genome shotgun sequencing done?

Answer 6

Whole genome shotgun sequencing involves:

1. cut many genome copies into randome fragments.
2. sequence each fragment.
3. overlap sequence reads to develop a contig (continuous sequence).
4. overlap contigs for complete sequence.

This can leave gaps which can be filled in later via targeted sequencing.